COMPOSITION FOR ACCELERATING WOUND HEALING

TECHNICAL FIELD

[01] The present disclosure relates to compositions that accelerate wound healing, and more particularly to compositions that may be applied directly over the skin to accelerate wound healing in mammals.

BACKGROUND

[02] Chitosan (poly(N-acetylglycosamine)) is partially deacetylated chitin which is one of the most abundant polysaccharides in nature second only to cellulose. It has a sugar backbone consists of b-1,4 linked glucosamine with a high degree of N-acetylation (70-90% N-acetylglucosamine and 10-30% D-glucose units), a structure very similar to that of cellulose; the only difference being the replacement of the hydroxyl by amino groups. Chitosan would be one of the most interesting polymers that has useful effects in all stages of wound healing including acceleration of healing, stimulation of immune response, antimicrobial action, haemostatic action and management of exudates; however, Chitosan oligosaccharides may cause cytotoxicity if absorbed.

[03] Vitamins play important roles in the metabolism, immunity, and food digestion. Certain vitamins such as B vitamins are known to play specific role in wound related metabolism and tissue repair. Biotin is an interesting B vitamin that possesses a role in converting glucose to ATP, which is an important process in producing the necessary energy required for wound healing. Biotin was introduced orally and topically for the treatment of wounds in the literature.

[04] Attempts to develop compositions and formulations containing Chitosan and Biotin were disclosed in the art; however, those attempts included utilization of several chemical mediators and/or organic solvents to synthesize final products, in which Chitosan was covalently bonded to Biotin. SUMMARY

[05] Aspects of the present disclosure provide a composition for accelerating wound healing, including Chitosan ions bonded to Biotin ions, which may result by means of ionic bonding.

[06] In aspects of the present disclosure, Chitosan ions may have a high molecular weight.

[07] In some aspects, Chitosan ions may have a molecular weight of at least 400,000 Da.

[08] In some aspects, the composition may include about 20%-80% of Biotin ions by volume.

[09] In other aspects, the composition may include about 20% of Biotin ions by volume.

[010] Aspects of the present disclosure further provide a method for producing a Chitosan-Biotin composition for accelerating wound healing, including Chitosan ions bonded to Biotin ions, wherein the method may include mixing Biotin with an alkalinizing agent at a molar ratio of about 1 : 1, dissolving about 20 ml of 1% Chitosan in an acidifying agent of about 0.1 M concentration, sonicating the mixture of Biotin and alkalinizing agent, mixing about 5 ml of Biotin- alkalinizing agent solution with the dissolved Chitosan solution, and drying the solution.

[Oil] Other aspects of the present disclosure provide a pharmaceutical composition including a composition, and a pharmaceutically acceptable carrier and/or excipient.

[012] In some aspects, the pharmaceutical composition is in oral dosage form.

[013] In some aspects, the oral dosage form may include liquid oral dosage form.

[014] In yet some aspects, the liquid oral dosage form may include emulsion, solution, suspension, syrup, and elixir.

[015] In other aspects, the oral dosage form may include solid oral dosage form.

[016] In yet other aspects, the solid oral dosage form may include a tablet, coated tablet, powder, powder for reconstitution, pellets, beads, mini-tablet, multilayer tablet, bilayered tablet, tablet-in tablet, pill, micro-pellet, small tablet unit, multiple unit pellet system, disintegrating tablet, dispersible tablet, granules, microspheres, multiparticulates, capsule, sachet, and sprinkles. [017] In yet other aspects, the pharmaceutical composition may be in topical dosage form.

[018] In other aspects, the topical dosage form may include powder, ointment, film, cream, lotion, suppository, gel, spray, and drops.

BRIEF DESCRIPTION OF THE DRAWINGS

[019] The disclosure will now be described with reference to the accompanying drawings, which illustrate embodiments of the present disclosure, without limiting the scope of the disclosure thereof, and in which:

[020] FIG. 1 illustrates chemical interaction between Chitosan and Biotin ions in embodiments of the present disclosure.

[021] FIG. 2 illustrates a flow diagram of a process of forming a Chitosan -Biotin composition, configured in accordance with embodiments of the present disclosure.

[022] FIG. 3 illustrates a plot comparing Fourier transform infra-red (“FTIR”) spectra of Chitosan, Biotin, and Chitosan-Biotin ionic composition prepared in accordance with

embodiments of the present disclosure, wherein“CSHC1” represents Chitosan,“BIO” represents Biotin, and“CS/BIO” represents Chitosan-Biotin composition.

[023] FIG. 4 illustrates a plot comparing a differential scanning calorimetry (“DSC”) of Chitosan, Biotin, and Chitosan-Biotin ionic composition prepared in accordance with

embodiments of the present disclosure, wherein“CSHC1” represents Chitosan,“BIO” represents biotin, and“CS/BIO” represents Chitosan-Biotin composition.

[024] FIG. 5 illustrates a plot comparing a thermal gravimetric analysis (“TGA”) of Chitosan, biotin, and Chitosan-Biotin ionic composition prepared in accordance with embodiments of the present disclosure, wherein“CSHCF” represents Chitosan,“BIO” represents Biotin, and “CS/BIO” represents Chitosan-Biotin composition.

[025] FIG. 6 illustrates an atomic force microscopic (“AFM”) image of Chitosan-Biotin ionic composition prepared in accordance with embodiments of the present disclosure, wherein the image scale is 50 pm. [026] FIG. 7 illustrates an AFM image of Chitosan, wherein the image scale is 50 mih.

[027] FIG. 8 illustrates a force modulation microscopy (“FMM”) image of Chitosan-Biotin ionic composition prepared in accordance with embodiments of the present disclosure, wherein the image scale is 50 pm.

[028] FIG. 9 illustrates an FMM image of Chitosan, wherein the image scale is 50 pm.

[029] FIG. 10 illustrates a column chart comparing wound contraction levels in mice after applying nothing, Chitosan, and Chitosan-Biotin ionic composition prepared in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION

[030] Embodiments of the present disclosure provide a composition for accelerating wound healing in mammals, wherein such composition may include Chitosan and Biotin ions bonded to each other by means of ionic bonds. FIG. 1 illustrates a chemical structure of a Chitosan-Biotin ionic composition for use in healing wounds, configured in accordance with embodiments of the present disclosure.

[031] As illustrated in FIG. 1 Chitosan cations may bind to Biotin anions forming ionic bonds between such two molecules, wherein such type of bonding may cause Biotin ions to be slowly released from the composition due to slow dissociation of Biotin ions from Chitosan ions.

[032] In embodiments of the present disclosure, Chitosan cations may have a molecular weight of about 400,000 Da or more.

[033] In embodiments of the present disclosure, the ratio of Biotin ions to Chitosan ions may range from about 20% by volume to about 80% by volume.

[034] In some embodiments, the ratio of Biotin ions to Chitosan ions may be about 20% by volume.

[035] Chitosan ions in embodiments of the present disclosure may be obtained from any of Chitosan salts and/or derivatives, which may include Chitosan HC1, Chitosan carbonate, Chitosan acetate, Chitosan sulfate, Chitosan phosphate, N-trimethyl chitosan, quaternary ammonium chitosan, or any combination thereof.

[036] Biotin ions in embodiments of the present disclosure may be obtained from any of Biotin salts and/or derivatives, which may include Biotin sodium, Biotin potassium, Biotin calcium, Biotin magnesium, or any combination thereof.

[037] Referring to FIG. 2, in embodiments of the present disclosure, the Chitosan-Biotin composition the composition may be formed by (a) mixing Biotin with an alkalinizing agent (e.g., sodium bicarbonate at 1 : 1 molar ratio) (process block 2-1); (b) dissolving about 20 ml 1% Chitosan in an acidifying agent (e.g., HC1 of about 0.1 M concentration) (process block 2-2); (c) sonicating the mixture (e.g., by a probe sonicator for about 10 minutes) (process block 2-3); (d) mixing about 5 ml of Biotin- alkalinizing agent solution with about 20 ml of dissolved Chitosan solution to produce a mixed solution with a pH of about 5±0.5 (process block 2-4); and (e) drying the mixed solution (e.g., in an oven at about 50°C for about 24 hours) (process block 2-5).

[038] Embodiments of the present disclosure further provide a pharmaceutical composition including a Chitosan-Biotin ionic composition, and an acceptable carrier and/or excipient.

[039] The term“pharmaceutical acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; binding agents such as hypromellose; disintegrating agents such as crosscarmellose; water; malt; gelatin; talc; thickening agents such as hydroxypropyl methyl cellulose; Hydroxy propyl cellulose; Ethyl cellulose; non-ionic surfactants such as polysorbates; sorbitan esters; excipients such as cocoa butter and suppository waxes; oils such as peanut oil; cottonseed oil; safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[040] The term“pharmaceutically acceptable” means at least non-toxic. The therapeutically active component may be present in the above-mentioned pharmaceutical composition, with a concentration of about 0.1 to 99.5% by weight, or maybe about 0.5 to 95% by weight of the total mixture.

[041] The pharmaceutical composition can be, for example, in a liquid form, e.g. a solution, syrup, emulsion, and suspension, or in a solid form, e.g., a capsule, caplet, tablet, pill, powder, and suppository. Granules, semi-solid forms, and gel caps are also considered. In case that the pharmaceutical composition is a liquid or a powder, dosage unit optionally is to be measured, e.g., in the dosage unit of a teaspoon.

[042] The pharmaceutical composition of this disclosure can be formulated for oral administration in solid or liquid form, or for topical administration. The pharmaceutical composition can be administered to humans and other mammals orally or topically (e.g., as a powder, ointment, film, cream, lotion, suppository, gel, spray, or drop).

[043] The disclosure is now further illustrated on the basis of Examples and a detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustrating and description only; they are not intended to be exhaustive or to limit the disclosure to the precise form disclosed.

Example 1

Preparation of Chitosan-Biotin composition Materials

[044] D-biotin (Vitamin H), CM S095-1-G, Min assay 98.5% and Chitosan (degree of deacetylation 87%, high molecular weight, viscosity 2% (w/v) in 0.1 M HC1 at 100 rpm, 60 Pa at 25°C) were commercially purchased from Himedia Laboratories Pvt, Mumbai 400086, India. Other reagents and chemicals were of analytical grade. Preparation Scheme

[045] Biotin was mixed with sodium bicarbonate (1 : 1 molar ratio) and sonicated using a probe sonicator (e.g., using a commercially available Branson Sonifier 250, Branson Ultrasonics, USA) for about 10 minutes till a clear solution was obtained. Then, about 5 ml of sodium bicarbonate/Biotin solution was vigorously mixed with about 20 ml of a 1% Chitosan (dissolved in about 0.1 M HC1) solution to produce a mixed solution with a pH of about 5±0.5. The solution was poured in a plastic Petri dish and left to dry in an oven at about 50°C for about 24 hours. Another Petri dish containing about 25 ml of 1% Chitosan (dissolved in 0.1 M HC1) was also dried and kept for comparison purposes.

Chitosan-Biotin Film Preparation

[046] For preparing a Chitosan-Biotin film, the preparation scheme described above was adopted. Different volume ratios of Biotin to Chitosan (4: 1, 1 : 1 and 1 :4) were prepared. The ratio (1 :4) was able to form a continuous film structure.

Example 2

Characterization of Chitosan-Biotin film

[047] Throughout this Example 2, reference will be made to FIGS. 3-9.

Fourier transform Infra-Red (“FTIR”]

[048] Small pieces of film samples of Chitosan, Chitosan-Biotin film and Biotin were crushed in the presence of KBr and compressed to form transparent disks. The disks were scanned using a FTIR spectrophotometer (e.g., using a commercially available IR Prestige 21, Shimadzu, Japan) in the following wave number range 500-2000 cm ¹ . FIG. 3 illustrates the FTIR spectra of samples used. As seen in FIG. 3, Chitosan has the bending bands of amine, asymmetric (N-H) at (1655 cm ¹ ) and symmetric (N-H) at (1540 cm ¹ ), while Biotin has a stretch band of (C=0) vibration of carboxylic acid at (1735 cm ¹ ). The stretch (C=0) band is shifted lower at (1712 cm ¹ ), compared to the stretch (C=0) vibration of a carboxylic acid (1734 cm ¹ ), consistent with the partial delocalization occurred in a carboxylate ion of Biotin. Two new peaks at (1712 cm ¹ ) and (1625 cm ¹ ) of Chitosan-Biotin are assigned to stretching (C=0) of carboxylate group and bending of (N-H) of ammonium ion, respectively. This suggested that Biotin and Chitosan may develop ionic interaction.

Differential scanning calorimeter (“DSC”) and Thermogravimetric analysis (“TGA”]

[049] Simultaneous DSC and TGA scans were measured using a calibrated STA449 F3 Jupiter (commercially available from Netzsch, Germany). A scan rate of about 10°C/minute was used with a temperature range from about 25°C to about 350°C. A sample of about 5 mg was scanned in an open pan study. FIGS. 4 and 5 illustrate DSC and TGA plots of Chitosan, Chitosan-Biotin film, and Biotin, respectively. As can be seen from FIGS. 4 and 5, the reported melting point of Biotin was about 232°C to 233°C, and started to decompose at about 300°C. The decomposition of Chitosan appeared by exothermic peaks in the range of about 190°C to about 220°C. The ionic interaction between sodium Biotin and Chitosan has elevated the decomposition temperature to about 270°C, while the absence of a melting peak of Biotin may indicate an ionic interaction with Chitosan.

Atomic Force Microscope (“AFM”)

[050] Surface morphology of Chitosan and Chitosan-Biotin film prepared in accordance with embodiments of the present disclosure was scanned (e.g., using a commercially available AFM, Dimension Edge, Bruker Force Modulation, Model MPP-Z1100-10, Part # RFESP, Material 0.01- .025 Ohm-cm, Antimony (n) doped Si, Germany). The software for scanning was the commercially available NanoDrive V8.01, (Build Rl.65039) copyright 2010 Veeco Inc. FIGS. 6 and 7 illustrate AFM images of Chitosan and Chitosan-Biotin film configured in accordance with embodiments of the present disclosure, respectively. FIGS. 8 and 9 illustrate force modulation microscopy (“FMM”) images of Chitosan and Chitosan-Biotin film configured in accordance with embodiments of the present disclosure, respectively. As can be seen in FIGS. 6, 7, 8, and 9, the Chitosan film produced a smooth surface with regions of folding due to water evaporation during sample drying, while the Chitosan-Biotin film surface was rough indicating the formation of a composite film. The differences between both the rough and smooth surfaces may be related to the difference in physicochemical properties of the Chitosan-Biotin film compared to that of the Chitosan film. Example 3

Evaluation of Chitosan-Biotin film for wound treatment Animals

[051] Animals, male albino Swiss mice (25-30 g), were purchased from King Abdulazziz University, Jeddah. The animals were acclimatized for two weeks with free access to water and food prior and during the conduction of the experiments. The animal studies were conducted in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC).

Film Mucoadhesion Test

[052] Three dry film samples were prepared from Chitosan (prepared in 0.1 M HC1, pH from about 3.5 to about 4 for 1% solution), partially neutralized Chitosan using 0.1 M NaOH to produce a pH of about 5.5 and Chitosan-Biotin ionic complex (1% dispersion in water, pH about 5.5) via pouring 1% solution of each sample in plastic Petri dishes (of about 25 ml) and left to dry in an oven at about 70°C for about 24 hours.

[053] A freshly prepared dorsal mouse skin with rectangle size of about 5 x 45 mm, same size, put each film with thickness of about 0.1 mm of same skin size to the mucosal side and fix the side on a hard surface of wood in a tilted position with an angle of about 70° and exposed the film to falling water with a flow rate of about 150 ml/min, and the distance between the tip of the skin and water source was about 60 mm in height. The detachment time was recorded for the Chitosan- Biotin film and the Chitosan film. The skin was wetted before attaching the films gently to the surface of the skin and let to withstand for about 15 seconds prior to water flow. The detachment (adhesion) time was 0.50±0.30, 13.83±1.04, and 25.33±2.52 minutes for the Chitosan film, the Chitosan-Biotin film, and the partially neutralized Chitosan, respectively. The Chitosan sample was dissolved very rapidly (i.e., the film disappeared completely within around half minute), while the partially neutralized Chitosan and Chitosan-Biotin films were detached as intact films from the skin surface at specific time. The partially neutralized Chitosan showed a significantly higher adhesion time (p<0.5) than that of the Chitosan-Biotin film. Chitosan is a fully ionized water- soluble polymer, while neutralization of the protons produces a less water soluble polymer with partial ionization. The ionized Chitosan dissolves easily in water, while the partially neutralized Chitosan has a much lower water solubility, which keeps it as an intact film in water. In the case of the Chitosan-Biotin ionic complex, the presence of hydrophobic functional groups in Biotin could enhance the water-insoluble property of the ionic complex and decrease the attachment to the skin mucosal side, which encouraged its earlier detachment compared to the partially neutralized Chitosan.

Excision wound model

[054] The mice were divided into three groups each of six animals. After induction of anesthesia with intramuscular dose of about 100 mg/kg of 10% ketamine, the dorsal skin of the mice was shaved then using the excisional wound model. The epidermal, dermal, hypodermal, and panniculus carnosus layers were completely removed from a predetermined region forming a 7 mm diameter circular wound area. Group 1 wounds were treated by placing a plane film of Chitosan on the wound having the same size of the wound. Group 2 wounds were treated with a film of Chitosan-Biotin prepared in accordance with embodiments of the present disclosure. Group 3 wounds were left uncovered (i.e., the untreated control group) to the open environment, and the animals were individually kept in separate cages. The changes in wound area were measured in millimeters using a Caliper every three-day interval. The decrease in the wound size was monitored based on the percent wound contraction according to the following formula:

_, _ (Wound area on day 0 - Wound area on day n)

Wound contraction (%) = - - - - - x 100%

Wound area on day 0 wherein n represents day number after treatment.

[055] As illustrated in FIG. 10, the groups of animals that received Chitosan and Chitosan-Biotin films showed significant wound contraction at all time intervals compared to the control group (p<0.05). Upon comparing the Chitosan film with the Chitosan-Biotin film, at the beginning (i.e., days 3 and 6), the Chitosan film showed a higher percent of contraction, then later did the Chitosan- Biotin film. This could be attributed to the fact that Chitosan can initially form a moist gel on the surface of the wound due to its water solubility, while Chitosan-Biotin is a water-insoluble composition. It was reported that the wetter wounds showed a much faster healing rate than the less moist ones. Further, Chitosan may be solubilized and absorbed through the epidermis and exerted its biological activities that help faster wound healing. On the contrary, the Chitosan- Biotin film can act as a sustained release composition for both the Chitosan and the Biotin due to the low water-solubility as a complex. Thus, its action may take a longer time, but it has some synergistic action in wound healing as observed later.

Skin elasticity test

[056] Skin elasticity was measured using a thermomechanical analyzer (e.g., using a commercially available TMA 402 FI hyperion, Netzsch Thermal Analysis, Germany) at isothermal conditions (32+0.5°C) where a constant force of about 3 N was exerted on rectangular healed dorsal skin mice samples. The mice were euthanized, and the skin samples were excised after eighteen days of treatment with Chitosan film, Chitosan-Biotin film, and a control group (i.e., with no treatment). The skin elongation ( A L I L _g ) was measured by tension mode by applying a constant force for about 5 minutes. A slope of the linear portion starting from the period of about 2 minutes to about 5 minutes was used for each sample as an indicative for the skin elasticity rate constant (SEC, ( L / L _g )/min). Such slope indicates skin elongation while applying a constant force on a portion of healed skin after injury, and therefore is an indicative measure of skin elasticity after treatment.

[057] Table 1 illustrates the healed skin elasticity determined by TM for mouse skin after eighteen days of treatment with Chitosan-Biotin and Chitosan films compared to control skin groups.

Table 1

Control skin after

Chitosan-Biotin Chitosan Control intact skin wound healing

with no treatment (untreated)

Skin

0.167±0.0482 0.0805±0.0111 0.059+0.010 0.360+0.0487 elasticity

(ratio/min)

Coefficient >0.99 >0.98 >0.98 >0.99

( R ² ) [058] As shown in Table 1, the elasticity of the formed skin after wound healing was much different from that of the intact skin. Healing occurred via filling of the wound area by granulation tissue, where tissues containing collagen played an important role in maintaining skin integrity and elasticity. It was reported that after fourteen days of wound maturation scar formation usually happened. The scarred skin formed upon wound healing was found to be stiff, rough, dry, and inflexible compared to the intact skin. The skin treated with the Chitosan-Biotin film configured in accordance with embodiments of the present disclosure produced more elastic skin compared to that of the Chitosan film, which could be justified due to the increase in healing rate due to the presence of the Biotin during wound healing process.

[059] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.

[060] In describing and claiming the present invention, the following terminology will be used.

[061] The singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise.

[062] As used herein with respect to an identified property or circumstance,“substantially” refers to a degree of deviation that is sufficiently small so as to not measurably detract from the identified property or circumstance. The exact degree of deviation allowable may in some cases depend on the specific context.

[063] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a defacto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[064] Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly recited limits of 1 to approximately 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as“less than approximately 4.5,” which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

[065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[066] Following long-standing patent law convention, the terms“a” and“an” mean“one or more” when used in this application, including the claims.

[067] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[068] As used herein, the term“about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method. [069] As used herein,“significance” or“significant” relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is“significant” or has“significance,” statistical manipulations of the data can be performed to calculate a probability, expressed as a“p value.” Those p values that fall below a user-defined cutoff point are regarded as significant. In some embodiments, a p value less than or equal to 0.05, in some embodiments less than 0.01, in some embodiments less than 0.005, and in some embodiments less than 0.001, are regarded as significant. Accordingly, a p value greater than or equal to 0.05 is considered not significant.

[070] As used herein, the term“and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase“A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.