FIELD OF THE INVENTION

The present invention relates to tunicate extracts and uses thereof in wound healing. More specifically, the invention relates to tunicate extracts and their use in enhancing wound healing.

BACKGROUND OF THE INVENTION

The healing of skin wounds is a multiphasic process requiring the coordinated effort of many cell types, clotting factors and growth factors. The complex process is comprised of three main phases (inflammatory, proliferative, and remodelling), each of which has its own specific events. Perturbation of any of these phases causes chronic wound exposure, infection and scarring.

In the initial stage, characterized by acute and controlled inflammation, vasoconstriction and platelet aggregation activate clotting factors to prevent excessive blood loss. At the same time, epidermal cells such as keratinocytes migrate to initiate wound closure. In this early phase, production of inflammatory mediators and growth factors is essential to the proper formation of the early scar tissue.

Wound treatments have essentially remained unchanged for centuries and involve three basic approaches: rehydration, oxygenation and prevention of infection. The type of wound naturally dictates the type of clinical approach to its treatment, but there are many similarities in therapies. Wounds occurring on mucous membranes, such as the cornea, are often treated with antibiotic liquids designed to fight infection and hydrate the wounds ^1"6 . Deep tissue injury involving the tearing of tissues and ligaments is often treated with surgery or physiotherapy ⁷ . Pressure ulcers can be treated with topical phenytoin, silver preparations, and growth factors, but clinical studies are still undecided as to their efficacy ⁸ . Cutaneous wounds are often treated with negative pressure therapy which is believed to increase vascular flow, but may result in pain and ischemia if improperly administered ²¹ . Chronic non-healing cutaneous wounds are sometimes treated with mesenchymal stem cell therapy ⁹ . All of these therapies are expensive and carry significant risks and side effects for the patient. Advancements in material sciences has created fibrous synthetics of nylon, polyethylene, polypropylene, and polyvinyls which are placed on top of the wound and significantly accelerate the natural wound healing process ^{10, 11} . Wet polymer wound dressings are used to form a physical shield as well as source of hydration for the wound. Tissue engineering has given rise to "living skin equivalents" that serve as sophisticated wound dressings to temporarily protect and hydrate the wound, but may also increase the production of growth factors essential for proper wound healing ¹² . All of these methods are mainly mechanistic in their approach - that is, they do not target growth factor generation and epithelial cell proliferation per se. In order to further accelerate wound closure and promote tissue remodelling (to prevent scar formation), a more sophisticated approach that directly promotes growth factor expression is required.

SUMMARY OF THE INVENTION

The present invention relates to tunicate extracts and uses thereof in wound healing. More specifically, the invention relates to tunicate extracts and their use in enhancing wound healing.

The present invention provides a method of preparing a tunicate water extract comprising: a) extracting a Styela clava tunicate sample with hot water; and

b) collecting the tunicate water extract obtained in step a).

In the method as just described, the tunicate sample may be lyophilized prior to step a); the lyophilized sample may optionally be milled prior to step a). The methods as just described may optionally comprise sequentially extracting the tunicate sample with hexane, acetone, methanol, and hot water in step a).

In the method as described above, the water extract may be further processed by ethanol precipitation, chromatography, ultrafiltration, desalting, drying by rotatory evaporator, centrifugal vacuum evaporator and/or spray dryer, or any combination thereof. Optionally, the water extract may be further fractionated by size exclusion chromatography, ion exchange chromatography, and/or papain hydrolysis followed by precipitation at basic pH, yielding sub- fractions. In turn, the sub-fractions may be further processed by ethanol precipitation, chromatography, ultrafiltration, desalting, drying by rotatory evaporator, centrifugal vacuum evaporator and/or spray dryer, or any combination thereof.

The present invention also provides a tunicate water extract obtained from Styela clava. The tunicate water extract may be obtained by any method as described herein. The tunicate water extract, fraction, or sub-fraction thereof of the present invention may be characterized by any of the proton NMR spectrum of Figure 4. The tunicate water extract, fraction, or sub- fraction thereof of the present invention may also comprise acetamide-substituted polysaccharides or protein glycans as the main components. The present invention further provides a method of enhancing wound healing comprising contacting a wound with one or more than one of the tunicate water extract, fraction, or sub- fraction thereof described herein, or any mix of compounds or single compounds obtained therefrom. Additionally, there is provided a method of inducing production of VEGF and reducing constitutive production of IGF-β, IL-6, and TGF-β in wound healing, comprising contacting a wound with one or more than one of the tunicate water extract, fraction, or sub- fraction thereof described herein, any mix of compounds or single compounds obtained therefrom, or any combination thereof. The extract may be provided as a powder, liquid, or suspension, or as a component in a cream or emulsion, for applying directly to the wound, or as a coating for bandages, sutures or other medical equipment to aid in wound healing.

It is presently shown that the tunicate water extract designated PTC-1400 has significant wound healing activity (Example 2; Fig. 2). Specifically, PTC-1400 is effective at promoting wound closure within 9 hr of wounding. Additionally, PTC-1400 is shown to significant production of VEGF, and may reduce constitutive production of IGF-β, IL-6 and TGF- β, all of which may change the kinetics and physiology of wound closure. Thus, the water extract of Styela clava (Clubbed tunicate) may provide a natural, improved compound for enhancing would healing.

Additional aspects and advantages of the present invention will be apparent in view of the following description. The detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the scope of the invention will become apparent to those skilled in the art in light of the teachings of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:

FIGURE 1 shows proton NMR spectra for various extracts of the tunicate samples. FIGURE 1 A shows hexane extracts (1 - PTC-3100; 2- PTC-2100; 3- PTC-1 100); FIGURE 1 B shows acetone extracts (1 - PTC-3200; 2- PTC-2200; 3- PTC-1200); FIGURE 1 C shows methanol extracts (1 - PTC-3300; 2- PTC-2300; 3- PTC-1300); and FIGURE 1 D shows hot water extracts (1 - PTC-3400; 2- PTC-2400; 3- PTC-1400).

FIGURE 2 is a graph showing the effect of tunicate extracts on wound closure. Human keratinocytes were wounded and immediately treated with PTC-1 100 for indicated time points. A) PTC-1 100; B) PTC-1200; C) PTC-1300; D) PTC-1400; E) PTC-2100; F) PTC-2200; G) PTC-2300; H) PTC-2400; I) PTC-3100; J) PTC-3200; K) PTC-3300; and L) PTC-3400. Data are represented as mean ± SEM. N=4. * p<0.05 compared to a vehicle control.

FIGURE 3 shows bar graphs summarizing the effects of PTC-1400 on angiogenic factor production by human mast cells. Human mast cells were pre-treated with PTC-1400 then stimulated with substance P (SP). Production of specific angiogenic factors was measured using a protein array and compared to a standard curve for quantitation (n=3). Asterisk represents significant differences (p<0.01 ) when compared to untreated (untr) control. A) TNF;

B) VEGF; C) FGF-β; D) EGF; E) IGF-1 ; F) IL-6; G) IGF-β; and H) leptin. FIGURE 4 shows proton NMR spectra for the PTC-1400 (3) and its sub-fractions YW-T-4 (2) and YW-T-6 (1 ).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to tunicate extracts and uses thereof in wound healing. More specifically, the invention relates to tunicate extracts and their use in enhancing wound healing.

There is increasing interest in developing effective and safe natural health products that can be used as alternative treatment options or co-solutions in enhancing wound healing. The present invention provides methods and products as a means to attain this goal.

The present invention provides a method of preparing tunicate extracts comprising:

a) extracting a Styela clava tunicate sample with hot water; and

b) collecting the tunicate water extract obtained in step a).

The tunicate sample may be obtained or harvested from any suitable tunicate species. Tunicates, also referred to as "urochordates", are marine organisms belonging to a group of underwater filter feeders with incurrent and excurrent siphons. Of particular interest in the present invention are tunicate samples comprising species Styela clava. Tunicate samples may be harvested from coastal areas globally, including but not limited to the coastal areas of North America, Europe, Asia, and South America. For example and without wishing to be limiting in any manner, the tunicate sample may be obtained from coastal areas in eastern Canada or the north-eastern USA. The tunicate sample may be collected at any suitable time of year; for example, and without wishing to be limiting in any manner, the tunicate sample may be collected between about September and May. Alternatively, the tunicate samples may be obtained from cultured tunicates. The tunicates may be cultured in an appropriate environment; methods for cultivating tunicates would be known to those of skill in the art. Optionally, the cultivated tunicates may be bioengineered (via hybridization or genetic engineering) to produce extracts exhibiting increased bioactivity characteristics.

The tunicate sample is extracted with hot water. As would be understood by one of skill in the art, the tunicate sample may be extracted directly with hot water to obtain a water extract; alternatively, the tunicate sample may be sequentially extracted with various solvents, including hot water. For example, and without wishing to be limiting in any manner, the tunicate sample is sequentially extracted with hexane, acetone, methanol, and hot water. By the term "sequentially extracted", and according to this example, it is meant that the tunicate sample is first extracted with hexane, and the resulting solid residue is then extracted with acetone; the solid residue resulting from acetone extraction is then extracted using methanol; the resulting solid residue is subsequently extracted with hot water. The tunicate sample may be extracted with additional or different solvents, not limited to those included herein. As would also be understood by a person of skill in the art, the tunicate sample may be extracted in a different order than that listed herein.

For water extraction, the tunicate sample or the solid residue resulting from the methanol extraction may be mixed with hot water. The water may be at a temperature between about 30°C and 100°C; for example, and without wishing to be limiting, the water may be at about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C, or any temperature there between or any range of temperatures defined by these values. In a specific, non-limiting example, the water may be at about 80 to 90°C; in a further non-limiting example, the water may be at about 90°C. The water is mixed with the solid residue in an a ratio of about 1 :20 original sample weight:water, or in a ratio in the range of about 1 :5 to 1 : 100; in a non-limiting example, the ratio may be about 1 :5, 1 :10, 1 :15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1 :55, 1 :60, 1 :65, 1 :70, 1 :75, 1 :80, 1 :85, 1 :90, 1 :95, 1 : 100, or any ratio therebetween. In a specific, non-limiting example, 1 g of sample may be mixed with 20 ml_ water. The water extraction may proceed for any suitable time; for example and without wishing to be limiting, the water extraction may proceed for a time in the range of about 30min to 5hrs or more; for example, and without wishing to be limiting in any manner, the extraction time may be about 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, or 300 minutes or more, or any time therebetween. In a specific, non-limiting example, the extraction time may be about 300 minutes (5 hrs). As would be understood by one of skill in the art, the extraction time may vary based on the extraction temperature. The water extraction may also be done with the assistance of microwave, sonication, or other approaches facilitating extraction that are known in the art. Following extraction, the sample may be cooled then filtered using any suitable filtration method known in the art, such as paper filtration or vacuum filtration. Alternatively, centrifugation may also be used for separation of water extract from solid residue. Following filtration, the flow-through or supernatant fraction is collected and is labelled as the water extract. For optional hexane, acetone, and methanol extractions, the tunicate sample or the solid residue may be mixed with a suitable amount of the respective solvent. For example, and without wishing to be limiting in any manner, the amount of solvent may be in a ratio of about 1 :20 original sample weight.solvent, or in a ratio in the range of about 1 :5 to 1 :100; in a non- limiting example, the ratio may be about 1 :5, 1 :10, 1 :15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1 :55, 1 :60, 1 :65, 1 :70, 1 :75, 1 :80, 1 :85, 1 :90, 1 :95, 1 :100, or any ratio therebetween. In a specific, non-limiting example, 1 g of sample or solid residue may be mixed with 20 ml_ solvent. The extraction may proceed for any suitable time; for example, and without wishing to be limiting, the extraction time may be about 30 minutes, or for a time in the range of about 5min to 2hrs. In a non-limiting example, the extraction time may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120 minutes, or any time therebetween. The extraction may proceed at any suitable temperature; for example and without wishing to be limiting, the temperature may be about room temperature, or between about room temperature and the boiling point of the solvents used. The extraction may additionally incorporate any acceptable physical or mechanical method known in the art; for example, and without wishing to be limiting, the extraction may incorporate stirring and/or sonication. Additionally, the extraction may also include immersing and/or refluxing.

Following each extraction step, the sample mixture may be filtered using any suitable filtration method known in the art, such as paper filtration or vacuum filtration. Alternatively, separation of liquid extract and solid residue may be done by centrifugation. Following filtration or centrifugation, the flow-through or supernatant fraction is collected and is labelled as the respective extract, while the solid filtrate or precipitate (also referred to herein as the solid residue) is subjected to further extraction.

Prior to extraction, the fresh tunicate sample may be dried, lyophilized and/or homogenized, by any suitable method known in the art. For example, and without wishing to be limiting in any manner, the tunicate sample may be homogenized using a commercially available homogenizer, grinder, blender, etc. The tunicate sample may be dried or lyophilized, also referred to herein as "freeze-dried" using any suitable method of freeze-drying or other drying methods known in the art may be used, such as but not limited to oven drying, drum drying, or conveyor drying. The dry tunicate tissue may optionally be milled by any suitable method known in the art. For example, and without wishing to be limiting in any manner, the tunicate sample may be milled using a commercially available grinder, or manually ground (for example, using a mortar and pestle). Subsequent to milling, the sample may be immediately subjected to extraction, or may be stored. The sample may be stored under any suitable conditions, for example and without wishing to be limiting in any manner, the sample may be stored at -80°C to room temperature. In a specific, non-limiting example, the sample may be stored at -80°C, -20°C, 4°C, 10°C, 25°C, or room temperature.

The water extract resulting from the extraction method described herein may optionally be processed or refined further using any suitable method in the art; for example, and without wishing to be limiting in any manner, the water extract may be subjected to ethanol precipitation, chromatography, ultrafiltration, desalting, drying by rotatory evaporator, centrifugal vacuum evaporator and/or spray dryer, or any combination thereof. The water extract may optionally also be further fractionated. Such further fractionation may be accomplished using any suitable method known in the art, for example, but not limited to a method described herein, or the method as follows. In a non-limiting example, the water extract may be submitted to size exclusion chromatography and/or ion exchange chromatography, and/or papain hydrolysis followed by precipitation at basic pH or with EtOH, yielding soluble and insoluble sub-fractions of the water extract. Methods for size exclusion chromatography, ion exchange chromatography, papain hydrolysis, and basic or EtOH precipitation would be known to those of skill in the art. The sub-fractions may optionally be further processed by any suitable method known in the art, for example, but not limited to ethanol precipitation, chromatography, desalting, drying by rotatory evaporator, centrifugal vacuum evaporator and/or spray dryer, or any combination thereof. The present invention also provides a tunicate water extract, fraction, or sub-fraction thereof obtained from Styela clava. The tunicate water extract, fraction, or sub-fraction thereof may be obtained using the methods as described herein.

The tunicate water extract, fraction, or sub-fraction thereof of the present invention may be characterized by the proton NMR spectrum as shown in any of FIGURE 4 (1 ) to (3). The tunicate water extract, fraction, or sub-fraction thereof may further be described as comprising acetamide-substituted polysaccharides and protein glycans as the main components.

The present invention further provides a method of enhancing wound healing comprising contacting a wound with one or more than one of the tunicate water extract, fraction, or sub- fraction thereof as described herein, or any mix of compounds or single compounds obtained therefrom, or any combination thereof.

Also encompassed by the present invention is a method of inducing production of VEGF and reducing constitutive production of IGF-β, IL-6, and TGF-β in wound healing, comprising contacting a wound with one or more than one of the tunicate water extract, fraction, or sub- fraction thereof or any mix of compounds or single compounds obtained therefrom as described herein.

The extract described herein may be administered in an effective amount to obtain the desired effect described above. As would be known to those of skill in the art, a specific dosage will vary based on several factors such as age and body weight of the subject. For example, and without wishing to be limiting in any manner, an effective dosage may be approximately 0.7 - 2.1 grams, or any amount in the range described; in a specific, non-limiting example, the dosage may be 1 -2 g/day. Additionally, the extract may be provided as a powder, liquid, or suspension, or as a component in a cream or emulsion, for applying directly to the wound, or as a coating for bandages, sutures or other medical equipment to aid in wound healing.

It is presently shown that the tunicate water extract designated PTC-1400 has significant wound healing activity (Example 2; Fig. 2). Specifically, PTC-1400 is effective at promoting wound closure within 9 hr of wounding. Additionally, PTC-1400 is shown to significant production of VEGF, and may reduce constitutive production of IGF-β, IL-6 and TGF- β, all of which may change the kinetics and physiology of wound closure. Thus, the water extract of Styela clava (Clubbed tunicate) may provide a natural, improved compound for enhancing would healing.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.

Example 1: Preparation of tunicate extracts

Tunicate samples can be collected from coastal areas in eastern Canada (for example Nova- Scotia, New-Brunswick, Prince Edward Island, Newfoundland) or the northeastern USA (for example Maine, Massachusetts, Connecticut, Rhode Island, New York, New Jersey).

Three tunicate samples were obtained from PEI Aquaculture, Fisheries and Research Initiative Inc from different sites in PEI, in 2008: • 081006MB: Styela clava (Clubbed tunicate, collected in Malpeque Bay, PEI on Oct. 6, 2008);

• 081112BR: primarily Ciona intestinalis (Vase tunicate, collected in Brudenell River, PEI on Nov. 12, 2008),;

· 081203SH: a mixture of Botrylloides violaceus (Violet tunicate) and Botryllus schlosseri (Golden star tunicate), collected in Savage Harbour, PEI on Dec. 03, 2008.

The samples were processed, freeze-dried, milled, and kept at -80°C. The dry powder of each tunicate sample was extracted sequentially using four different solvents: hexane, acetone, MeOH, and hot water. The tunicate sample was mixed 1 :20 in hexane (1g in 20ml_ solvent), stirred for 30min at room temperature and then sonicated for 30min. The mixture was then filtered through filtration paper and the flow-through was collected. The extraction was repeated once and the solvent in the combined liquid extract was removed by rotatory evaporator then centrifugal evaporator (Genevap) to yield the hexane extract. The solid residue was then mixed 1 :20 (original sample weight) in acetone and stirred and then sonicated for 30min; the mixture was filtered and the flow-through was collected. Similarly, the extraction was repeated once and the solvent in the combined liquid extract evaporated (acetone extract). The solid residue was then resuspended in MeOH (1 :20), followed by 30min stirring and sonication; the mixture was then filtered and the flow-through was collected. The extraction was repeated once and the solvent in the combined liquid extract evaporated (MeOH extract). Finally, the solid residue was mixed with hot water (1 :20) and stirred at 80- 90°C for 5hrs. After cooling to room temperature and filtration, the flow-through was mixed with 3 volumes of 95% EtOH and put in ice bath for 2 hrs. The precipitate was then filtered, washed with EtOH, and collected by centrifugation. The solvent was removed from each extract by centrifugal evaporator (Genevap) and freeze-dryer to yield water extracts. Results of the extractions are shown in Table 1.

Table 1. Tunicate species and extracts

Sample

Sample Extract Extract Name Extract Mass % by mass

Mass

081006MB 40.204 Hexane PTC-1100 or MB-H 0.475 1.181

Acetone PTC-1200 or B- A 0.203 0.505

Methanol PTC-1300 or MB-M 8.509 21.165

Water PTC-1400 or MB-W 3.373 8.39

081 112BR 39.995 Hexane PTC-2100 or BR-H 0.726 1.815

Acetone PTC-2200 or BR-A 0.454 1.135

Methanol PTC-2300 or BR-M 11.582 28.959

Water PTC-2400 or BR-W 1.411 3.528

081203SH 40.015 Hexane PTC-3100 or SH-H 0.417 1.042 Acetone PTC-3200 or SH-A 0.257 0.642

Methanol PTC-3300 or SH-M 11.835 29.576

Water PTC-3400 or SH-W 1.72 4.298

Example 2: Proton NMR characterization of tunicate extracts

The extracts obtained in Example 1 were submitted to Proton NMR profiling. Briefly, 2 mg of the hexane, acetone, and MeOH extracts were dissolved in 100 μΙ_ of DMSO- d6, while 2 mg of water extracts were dissolved in 100 μΙ_ D ₂ 0. Sample solutions were transferred to 1.7 mm NMR tubes and proton NMR spectra were acquired on Bruker Bruker Avance III 600 MHz NMR spectrometer (Bruker Corporation, East Milton, ON) operating at 600.28 MHz ¹ H observation frequency and a temperature of 25±0.2°C. The signals were acquired, processed and analyzed using TopSpin ^® NMR data acquisition and processing Software (Bruker Biospin Ltd, East Milton, ON) integrated with the spectrometer.

Results in the form of NMR spectra are shown in Figure 1. General proton NMR profiling indicated that there is certain level of similarity in the main components of extracts prepared from different tunicate species. Hexane and acetone extracts comprised fatty acids (including polyunsaturated FAs), while the water extracts showed the presence of polysaccharides or protein glycans as the main components.

Example 3: Effect of tunicate extracts on wound healing

Human keratinocytes were used to test the effect of the tunicate extracts of Example 1 on healing of physically-induced wound. The effect on wound-healing was measured at different time points after the cells were incubated in the presence of each extract.

Human keratinocytes (A431 ; human skin epithelial cell line) were grown in minimal essential media (MEM with 10% fetal bovine serum, FBS) in a 24 hr plate to greater than 95% confluency. Media was replaced with M EM without FBS and cells were rested for 12 hr. The following day, a wound was formed using a small boar needle (19 gauge) scraped through the epithelial cell layer. This small wound was monitored by imaging software, which allowed for a more detailed and reproducible analysis of wound closure as a function of time. Conditioned media containing FBS (at 10% media volume) was used as a positive control. The negative control cells were grown in minimal medium (MEM without FBS) containing osmotically- controlled amino acids and salts without growth factors or clotting factors, and treated with vehicle (phosphate buffered saline, PBS). The tunicate extracts of Example 1 were dissolved in sterile PBS to a concentration of 25pg/mL The extract (10 ug/mL final concentration), positive control, or negative control was added to the wound immediately after formation. At time zero, each wound was analyzed using a Nikon digital camera at 100X magnification andimaging software; the wound area was calculated based on pixel numbers in the affected area using imaging Image J software. At 2, 6 and 9 hrs after initial wound formation, the wound area was again imaged and analyzed to determine pixel numbers within the affected area. Each wound area was normalized to the time 0 area and data is represented as a percent of initial wound area. Therefore, each wound is internally controlled for size and shape. Four independent trials were performed. Results of the assays are shown in Figures 2A through 2L and summarized in Table 2. Within 2 hours, the negative control wounds began to noticeably close, resulting in a reduction of wound area by approximately 20% (Fig. 2). However, at 2 hr, there was no statistical significance between the wounds treated with negative control (minimal medium) and positive control (serum growth factors and clotting factors). This was expected, since this early phase is unlikely to be regulated by growth factors and more likely to be dependent upon intrinsic keratinocyte cell cycle and normal proliferation. However, as factors released by damaged cells and cell debris (from wound formation) accumulated and activated nearby cells, wound closure kinetics changed. At 6 hours, the wounds treated with positive control media had closed approximately 60% of their area, whereas the wounds treated with the negative control media were significantly larger. By 9 hours, the difference between the wounds treated with positive and negative control media were significantly different in size, with the negative controls still displaying approximately 60% of their original wound area.

Table 2. Effect of tunicate extracts on wound closure 9 hr following application.

PTC-3300 79.89 1.9 -18.22 33.0 -123.0

PTC-3400 69.68 1.2 -8.01 22.8 -54.1 -

"+" denotes a positive effect on wound closure

"-" denotes a negative effect on wound closure

PTC-1100 did not significantly affect changes in wound area compared to the negative control, suggesting that PTC-1100 does not promote wound closure. Only one tunicate extract (PTC- 1400) was shown to have significant wound healing activity (Fig. 2 and Table 2). Specifically, PTC-1400 displayed wound healing activity as early as 6 hr after wound formation and was as effective as the positive control. Other tunicate extracts (PTC-2300, PTC-2400, PTC-3200, PTC-3300, and PTC-3400) analyzed were deleterious to wound closure and inhibited proper wound closure. For example, PTC-2300 inhibited wound closure; at 9 hours the wound area of wounds treated with PTC-2300 were larger than those treated with the negative control (Fig. 2 and Table 2).

Example 4: Analysis of tunicate extract effect on production of angiogenic factors

Since wound healing is dependent upon the tightly orchestrated production of growth factors required for accelerated cell migration and proliferation, the production of angiogenic factors by resident tissue cells (human mast cells) involved in tissue healing and remodelling was examined. PTC-1400 (obtained in Example 1 ), the extract that displayed the most potent wound healing activity (see Example 3) was chosen for these experiments. To quantify the production of angiogenic factors, a cytokine array was used. Substance P (SP), a neuropeptide known to be produced by peripheral nerves during wounding and involved in tissue remodelling during wound closure, was used as a positive control.

Human mast cells were suspended in media containing human recombinant stem cell factor (100 ng/mL) at a concentration of 1 x 10 ⁶ cells/ml. Cell suspensions were pre-treated with PTC-1400 (10 Mg/mL) for 1 hr then stimulated with substance P (SP; 1 Mg/mL) for 24 hr. Cell- free supematants were collected and stored at -80°C for up to three days. Angiogenic factor production was measured using an ELISA-based protein array and each analyte was compared to corresponding standard curve for quantification (n=3). Analytes tested were tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), fibroblast growth factor beta (FGF-β), epidermal growth factor (EGF), insulin growth factor I (IGF-I), interleukin-6 (IL-6), transforming growth factor beta (TGF-β) and lepin. Results are shown in Figure 3. Of the 8 different angiogenic factors analyzed (TNF, VEGF, FGF-β, EGF, IGF-I, IL-6, TGF-β, leptin), PTC-1400 induced significant production of VEGF and may potentiate the production of SP-induced IGF-I. There was also some indication that PTC- 1400 may reduce constitutive production of IGF-β, IL-6 and TGF- β, all of which may change the kinetics and physiology of wound closure.

VEGF is a known angiogenic factor that promotes the growth of new blood vessels to the regenerating tissue surrounding a wound and therefore production of VEGF is critical to the normal process of wound repair ¹³ . IGF-I promotes epithelial cell regeneration and wound closure ¹⁴ , but is only effective when produced locally (i.e., by tissue resident cells such as the mast cells tested herein) rather than systemically ^15"18 . Furthermore, IGF-l-mediated promotion of wound repair may be most significant in mucosal tissue injury such as gastric ulcers and respiratory tissue tears (associated with lung disease). In fact, impaired healing of chronic gastric ulcers in arthritic rats is, at least partly, accounted for by a decreased production of IGF-1. Since tunicate extracts induce IGF-I, these extracts may also improve the delayed healing of gastric ulcers in arthritic patients. Since IGF-I stimulates a high level of collagen formation by skin keratinocytes and fibroblasts, it also promotes the restoration of the extracellular matrix and thereby promote normal tissue regeneration ¹⁹ . This would be particularly relevant in burn wounds where extracellular matrix formation is aberrant ²⁰ .

Example 5: Further fractionation of tunicate water extracts

The PTC-1400 water extract of Example 1 was further fractionated and characterized by NMR.

In preparation, 250 mg PTC-1400 was desalted by washing with by washing with 70% EtOH then dried. The dried extract was subjected to papain hydrolysis in NaOAc, pH 6.8 for 18 hr. After deactivation at 95°C for 0.5 hr and cooling down, the precipitate was removed by centrifugation. The supernatant was mixed with 4 volumes of 70% EtOH, stored at 4°C, and the resulting supernatant (sub-fraction YW-T-4) and the precipitate (sub-fraction YW-T-6) were collected and freeze-dried (not desalted).

The PTC-1400 extract and its sub-fractions were then characterized by proton NM R, as described in Example 2. Results of the NMR characterization are show in Figure 4. As demonstrated in the spectra, water extract PTC-1400 and sub-fraction YW-T-6 contain polysaccharides (with acetamide substituents) and protein glycans as the main components, while sub-fraction YW-T-2 appears to have protein or peptides as main components.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution. REFERENCES

All patents, patent applications and publications referred to herein are hereby incorporated by reference in their entirety.

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