Isolated polysaccharide compounds and their uses and methods of production

Abstract

Described herein are isolated biological polysaccharide compounds. The biological polysaccharide compounds may be characterized by being isolated; having glycosyl linkages comprising 65-95% by weight 1:3 linked glucopyranosyl residues and 5-25% by weight 1:6 linked glucopyranosyl residues; a β-glucan purity of 85-100%; a molecular weight of 0.5-2.2 MDa; a TNF-α cytokine response in a human bioassay that is at least 1.5 times greater than the TNF-α cytokine response of a negative control in the human bioassay; and being essentially insoluble in aqueous solution. In at least one embodiment, a method of treating skin by topical application of a vehicle comprising the isolated biological polysaccharide to a skin site, such as a wound or burn, is described. Methods of manufacture are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT patent application of U.S. Utility Application No. 17 / 390,809, filed on July 30, 2021, with WIPO DAS code 8235, the specification of which is incorporated herein by reference.

[0002] Technical Field Described herein are isolated biological polysaccharide compounds, their methods of use and manufacture. More specifically, the polysaccharides may be highly purified forms of biologically derived polysaccharides, and the methods of use may relate to topical skin treatments. The methods of manufacture include additional manufacturing steps to conventional methods that appear to provide unexpected, clinically effective polysaccharide compounds. [Background technology]

[0003] Background technology Polysaccharide compounds and related aspects are described herein. For ease of explanation, the polysaccharides described herein are derived from yeast and may hereinafter be referred to interchangeably as biological polysaccharides, biologically derived polysaccharides, glucans, or β-glucans.

[0004] Biologically derived polysaccharides from yeast are known compounds composed of glycosyl bonds. Their use to promote wound repair, among other applications, is known. Non-cellulosic β-glucans are recognized as potent immune activators. β-glucans are generally safe and known to reduce post-operative infection rates.

[0005] Existing technology describes yeast cell membrane products as one of the key compounds in such products, among other findings for tissue repair, skin care, and other applications. The technology describes the use of yeast cell products for the treatment of skin disorders; a process for producing β-glucan from yeast; processing of yeast-derived skin cell products; revitalizing skin with topically applied particulate β-glucan from yeast; cereal-derived β-glucan as a gel or cream for the topical treatment of burns; preparation of small particle glucan using lyophilization; a process for extracting β-glucan using enzymes; use of carboxymethyl β-glucan in a carrier to treat skin after laser or chemical peel treatment; topical application of particulate β-glucan to treat skin after laser or chemical peel treatment; effects of purified yeast extract β-glucan with only 1-3 bonds and 1-6 side chains; and a gel glucan product from Saccharomyces cerevisiae.

[0006] There are gaps in the understanding of the complex relationship between the structure of β-glucans and their efficacy profile, as well as heterogeneity in approaches to clinical translation and to the extraction and purification of these agents. These gaps hinder the search for molecules that have actually demonstrated success in achieving clinically accepted endpoints set by regulatory authorities in double-blind, randomized, placebo-controlled trials.

[0007] To date, β-glucan has not received pharmaceutical or biological approval for the indication in wound healing. Despite prior art on molecules for use in wound healing, no formulation containing β-glucan has demonstrated clinical efficacy in double-blind, placebo-controlled human trials in venous leg ulcers or other chronic wounds around regulatory mandated clinical endpoints.

[0008] In the inventors' experience, the prior art does not address or describe how molecular characterization produces evidence of clinical efficacy. For example, the present technology does not explain what the branching structure of the glucan should be; what the molecular weight should be; what the ideal purity and measured immune response should be, etc., to achieve both regulatory approval combined with end product consistency and actual demonstrated clinical efficacy in wound healing endpoints.

[0009] Despite the vast amount of prior art in the field of glucans, no glucans have been approved to date that have met the valid endpoints of the U.S. Food and Drug Administration (FDA) in clinical trials. However, the need for effective and effective skin treatments remains, and effective new therapies would be of great value. It is noted that in the past 30 years, no drug has been approved with therapeutic claims to treat venous leg ulcers, despite the large unmet need demonstrated for this indication.

[0010] In particular, the inventors have determined that the prior art does not describe: -Accurate characterization and description of β-glucan compounds that demonstrate clinical efficacy on gold standard wound healing endpoints set by regulatory agencies in the treatment of venous leg ulcers and chronic wounds; -additional processing of the insoluble yeast (Saccharomyces cerevisiae) derived beta-glucan beyond that described in US 6,242,594; - a beta-glucan compound of higher purity than that described in US 6,242,594; -Particulate beta-glucan; -Unexpected demonstrated clinical efficacy in the treatment of cosmetic skin disorders and chronic wounds; - Use of a topically applied particulate biological polysaccharide compound to improve skin quality as measured by elastosis and wrinkles. - Uses biological polysaccharide compounds to provide clinical evidence of accelerated skin quality benefits following laser ablative laser surgery; -The use of biological polysaccharide compounds to initiate and further accelerate the healing of venous ulcers; -Demonstrated clinical efficacy in wound healing in a human double-blind, randomized, placebo-controlled study; - Proven clinical efficacy in accelerated skin quality as measured by clinical graded improvement in elastosis and wrinkles in a human double-blind, randomized, placebo-controlled study; -The technology also leads to moving away from insoluble biological polysaccharide compounds and insoluble β-glucan itself. In fact, the technology shows that insoluble materials should not be used in the local wound / skin environment because they are highly inflammatory and an excessive inflammatory response is harmful and actually worsens the healing outcome; -Evidence of significantly higher immune responses than prior art compounds for wound healing, producing unexpected positive effects; -A biological polysaccharide compound that has achieved positive Phase IIB data in the indication of venous leg ulcers and has achieved cosmetic endpoints acceptable to the FDA and other regulatory agencies to be granted a therapeutic label.

[0011] Further aspects and advantages of the biological polysaccharide compounds, their methods of use and preparation will become apparent from the following description, which is given by way of example only. Summary of the Invention

[0012] Described herein is an isolated, insoluble, biologically derived polysaccharide compound in a highly pure form.Methods of use of this compound are described, which show higher than expected levels of efficacy of this compound, particularly in topical skin treatment.A method of manufacture is also described, which shows alternative / additional manufacturing steps to conventional methods that appear to provide an unexpectedly effective polysaccharide compound.

[0013] In a first aspect, there is provided an isolated biological polysaccharide compound comprising one or more of the following: glucosidic linkages containing 65-95% by weight of 1:3 linked glucopyranosyl residues and 5-25% by weight of 1:6 linked glucopyranosyl residues; With β-glucan of 85-100% purity; with a molecular weight of 0.5-2.2 MDa; a TNF-α cytokine response in a human bioassay that is at least 1.5-fold greater than the TNF-α cytokine response of a negative control in a human bioassay; Including, It is essentially insoluble in aqueous solutions.

[0014] In a second aspect, there is provided a method of treating the skin of a patient in need thereof by topical application to a wound site of a vehicle, the vehicle comprising a therapeutically effective amount of an isolated biological polysaccharide compound substantially as described herein.

[0015] In a third aspect, there is provided the use of an isolated biological polysaccharide compound, substantially as described herein, in the manufacture of a medicament for the topical treatment of the skin of a patient in need thereof.

[0016] In a fourth aspect, Selecting the yeast cells; lysing the cells and collecting cell wall fragments; heating and acidifying the cell wall fragments to remove mannan and chitin; performing a phase separation using a solvent to remove additional mannan and additional chitin along with proteins, glycogen and lipids; separating the solvent and other non-polysaccharide compounds by boiling and drying; performing at least one water rinsing step after dissolution and before acidification; There is provided a method for producing an isolated biological polysaccharide compound substantially as described above, comprising:

[0017] The isolated biological polysaccharide compounds, methods of use and methods of manufacture described above have many advantages, one of which is that the compounds unexpectedly provide optimal immunogenic responses for topical treatment without adverse inflammatory effects.

[0018] Further aspects of the biological polysaccharide compounds, methods of use and methods for their manufacture will become apparent from the following description, given by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0019] [Figure 1A] The 1H NMR graph and the molecule are shown diagrammatically. The biological polysaccharides described are β-glucan-based polysaccharides with the general structure of (1->3)(1->6)-β-glucan. For simplicity, the key residues are labeled "BC", "Br", and "SC". BC represents main chain residues, Br represents branching residues, and SC represents side chain residues. [Figure 1B] The 1H NMR graph and the molecule are shown diagrammatically. The biological polysaccharides described are β-glucan-based polysaccharides with the general structure of (1->3)(1->6)-β-glucan. For simplicity, the key residues are labeled "BC", "Br", and "SC". BC represents main chain residues, Br represents branching residues, and SC represents side chain residues. [Diagram 2] Results of an Australian Phase I study to establish the safety and efficacy of a microparticulate form of the compound of the invention are presented. An open, uncontrolled study of six patients was conducted. The compound of the invention in vehicle was applied topically every 2-3 days for four weeks to patients where standard wound treatment had failed. No significant intolerance or toxicity was observed or reported in association with the use of the test article. Healing responses were observed in all six patients, with reductions in wound surface area ranging from 26% to 82% measured over 56 days. [Diagram 3]This presents the results of a Phase 2 I / II study, conducted to determine the efficacy of the compound of the present invention in a single-center, randomized, double-blind, vehicle-controlled study in 18 patients with CDVI ulcers refractory to standard wound management therapy. The compound of the present invention was compared to another form of glucan (Glucodine™) with a lower molecular weight range and a lower percentage of (1:6)-β-glucan side branches, and to vehicle (control). Efficacy was assessed by planimetry, measuring the surface area of ​​the wound. The mean improvement over 4 weeks was 4.4% in the vehicle group, 36.7% in the compound of the present invention group, and 17.3% in the Glucodine™ group. The results showed that the compound of the present invention contains a more effective active agent for promoting wound healing. [Figure 4] This paper presents the results of a Phase IIA study of a Phase 3 trial, conducted with the goal of identifying a suitable formulation on which to base a then-definitive regulatory Phase III study. This was a Phase 2, double-blind, randomized, vehicle-controlled study in which patients were assigned to one of three treatment groups based on randomization and using a computer-generated allocation sequence. Fifty-eight patients with chronic venous ulcers were recruited at two centers and randomly assigned to either high-dose active (1.0% compound of the invention gel) or low-dose active (0.1% compound of the invention gel), or gel base alone (vehicle control). The study was intended to provide a statistical evaluation of the efficacy and safety of the compound of the invention in patients with chronic venous insufficiency ulcers of the lower extremities. Data from the Phase II study demonstrated that the compound of the invention accelerated the rate of wound healing, with treated ulcers healing at a statistically significant rate of improvement compared to placebo-treated ulcers. The compound of the invention demonstrated a mean wound area reduction of 55% to 59% versus 10% for placebo. [Diagram 5]The results of a Phase IIA clinical trial of 26 patients after fractionated cosmetic laser treatment with gel containing the compound of the present invention are shown. The study showed that wound healing time was improved by an average of 30%. The efficacy results of the time to complete wound closure were faster when the compound of the present invention group was compared with the placebo group (labeled GLYC-101 0.1% and GLYC-101 1.0%) (p=0.0062 and 0.0331, respectively). [Figure 6-8] FIG. 1 shows photographs of an atypical example of a patient who experienced a reduction in wound size of a venous ulcer wound in a completed Phase IIA Australian clinical trial using a compound of the present invention. [Figure 9] The raw incidence of complete closure between the vehicle and active groups of the compound of the invention in a US-based Phase IIB trial of 82 patients is shown, along with the Phase III data and raw incidence difference of the existing product Apiligraf™. Apiligraf™ (see www.apiligraf.com) is supposedly the gold standard for venous leg ulcer (VLU) closure, reimbursing approximately US$1200 per application, and the usual treatment of VLUs with Apiligraf™ may require 3-15 applications. The compound of the invention achieved an adjusted 21% difference in complete closure incidence compared to Apiligraf®'s 17% complete closure incidence. Meaningful differences between the ITT and PP groups are noted. Given the differences in inclusion and exclusion criteria in the trial design (including blinding), the results are not strictly comparable, but provide a strong signal that the compound of the invention has comparable or superior efficacy to the gold standard product. [Figure 10]ODDS RATIO ("OR") output from an 82 patient Phase IIB trial in venous leg ulcers. It shows the OR of achieving 100% healing for the active group over placebo. The output is from a logistic regression adjusted for covariates known to affect healing. Interpretation of the OR, e.g., an OR of 1.4 means that the odds of healing in the TR987 group are 1.4 times higher than the odds of healing in the placebo group. Values ​​greater than 1 favor treatment and values ​​less than 1 favor placebo. Odds ratios for all groups are clinically meaningful (2.0 or greater), all favoring the compound of the invention, suggesting that the odds of achieving complete healing are twice as high for the active group versus placebo. [Figure 11] A comparison of the mean wound area reduction for patients who completed a Phase IIA clinical trial and a Phase IIB clinical trial is shown. This data demonstrates that compounds of the present invention produce consistent efficacy as measured by wound area reduction in two independent Phase II clinical trials. [Figure 12] Figure 1 shows the relationship between the efficacy of the compound of the invention and Apiligraf™ in treating venous leg ulcers. This shows the difference in incidence of complete closure vs. placebo. In this figure, the compound of the invention is labeled TR-987. This figure shows the difference in the proportion of patients who achieved complete closure between active and placebo. The compound of the invention has an adjusted difference in incidence of complete closure of 21% compared to an adjusted incidence of complete closure of 17% for Apiligraf™, showing the superior differentiation of the compound of the invention over the arguable gold standard product. [Figure 13] A comparison of the efficacy of the compounds of the present invention compared to Epifix™, a product derived from human placenta and approved as a human tissue healing product, using the indicator of wound area reduction (see https: / / mimedx.com / epifix / ). [Figure 14] 1 shows photographs of a representative example of wound size reduction in a patient with a venous ulcer wound in a completed Phase IIB 82 patient trial using a compound of the present invention. [Figure 15] 1 shows photographs of a representative example of wound size reduction in a patient with a venous ulcer wound in a completed Phase IIB 82 patient trial using a compound of the present invention. [Figure 16] 1 shows photographs of a representative example of wound size reduction in a patient with a venous ulcer wound in a completed Phase IIB 82 patient trial using a compound of the present invention. [Figure 17] An atypical example of a patient receiving a compound of the invention following a minimally invasive cosmetic procedure (CO2 fractionated laser treatment of the chest) and an atypical example of improved skin quality outcomes found in a Phase IIB laser ablation study, n=40. Patients using a compound of the invention in a Phase IIB trial had nearly double the incidence of enhanced skin quality as measured by elastosis and wrinkles at 28 days after use of a compound of the invention in a gel vehicle compared to placebo gel + standard of care (p<0.04, n=40). [Figure 18] The percentage of patients in the compound of the invention group and the vehicle group, respectively, who achieved a ≥ 1 point improvement in wrinkle score between baseline and day 28 (Fitzpatrick-Goldman classification) are shown. 85% of respondents achieved a wrinkle score of ≥ 1 for the compound of the invention group compared to the placebo group (only 50% of respondents achieved a wrinkle score of ≥ 1). P<0.04 for 70% of variances using chi-square or Fisher exact value. [Figure 19] The percentage of patients who achieved an improvement in elastosis score of ≥ 3 points between baseline and day 28 in the compound of the invention group and the vehicle group, respectively, is shown (Fitzpatrick-Goldman classification). Chi-square test for unadjusted proportions was used to determine significance, expressed as p-value. At day 28, 75% of respondents in the active group achieved an improvement score in elastosis of ≥ 3, compared with only 35% of respondents in the placebo group (114% variance P<0.011). [Figure 20]The efficacy of the compound of the present invention against a further technical product called Woulgan™, which the inventors believe to be the most relevant β-glucan patent publication for wound healing, is shown. Woulgan™ produced an 8.9-fold immune response compared to the control, as measured in an assay measuring TNFα response from human harvested macrophage cells. In contrast, the compound of the present invention (labeled TR-987) produced a 25.4-fold response compared to the same vehicle control. This means that the compound of the present invention is approximately 3 times more potent than Woulgan™ gel and is much more effective than conventional compounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] As mentioned above, described herein is an isolated biologically derived polysaccharide compound in a highly pure form.Methods of using this compound are described, and the unexpected effectiveness of this compound is shown, particularly in topical skin treatment.A method of manufacture is also described, which shows alternative / additional manufacturing steps to conventional methods that may provide an unexpectedly effective polysaccharide compound.

[0021] For purposes of this specification, the terms "about" or "approximately" and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, degree, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0022] The term "substantially" or grammatical variations thereof refers to at least about 50%, for example 75%, 85%, 95% or 98%.

[0023] The term "comprise" and grammatical variations thereof are intended to have an inclusive meaning that is to be interpreted to mean including not only the directly referenced recited component, but also other non-specific components or elements.

[0024] As used herein, the term "polysaccharide compound" and grammatical variations thereof include yeast-derived compounds containing glycosyl bonds. The term "polysaccharide compound" may hereafter be used interchangeably with the terms "biological polysaccharides," "biologically derived polysaccharides," "glucans," or "β-glucans." Unless otherwise specified, reference to one term should not be considered as limiting the use of the other term.

[0025] The term β-glucan or grammatical variations thereof is used herein to encompass or be a β-D-glucan.

[0026] Isolated biological polysaccharide compounds In a first aspect, there is provided an isolated biological polysaccharide compound comprising one or more of the following: glucosidic linkages containing 65-95% by weight of 1:3 linked glucopyranosyl residues and 5-25% by weight of 1:6 linked glucopyranosyl residues; With β-glucan of 85-100% purity; with a molecular weight of 0.5-2.2 MDa; a TNF-α cytokine response in a human bioassay that is at least 1.5-fold greater than the TNF-α cytokine response of a negative control in a human bioassay; Including, It is essentially insoluble in aqueous solutions.

[0027] Isolation In the context of this specification, the term isolated refers to a biological polysaccharide compound that has been isolated in a purified form from its native state, such as a cell or cell wall, etc. The exact isolation method may vary, but may include various steps for washing and removing impurities such as proteins and lipids.

[0028] The isolated biological polysaccharide compound may be substantially intact, i.e., the isolation process minimizes or avoids cleavage of chemical bonds within the compound, and the complexity and size of the biological polysaccharide compound remain intact. The complexity in this case may be particularly relevant for the 1:3 to 1:6 ratio of branched side chains of the biological saccharide compound.

[0029] biological polysaccharide compounds As noted above, isolated biological polysaccharide compounds are described herein.

[0030] In at least one embodiment, the biological polysaccharide compound described is derived from yeast. The biological polysaccharide compound may be derived from a yeast cell. The yeast cell may be derived from the species Saccharomyces cerevisiae.

[0031] The biological polysaccharide compound may be a glucan compound. The biological polysaccharide compound may be a β-glucan compound.

[0032] Insoluble The biological polysaccharide compounds are insoluble in aqueous solutions.

[0033] Solubility can be defined as the maximum concentration of a substance that can be completely dissolved in a given solvent at a given temperature and pressure. The solubility of a substance can be described in a variety of ways. USP / NF generally expresses solubility in terms of the volume of solvent required to dissolve one gram of drug at a particular temperature. Using this scale, the described biological polysaccharide compounds are practically insoluble or insoluble, with more than 10,000 parts of solvent required for one part of solute.

[0034] As can be seen from the manufacturing methods further described below, the biological polysaccharide compounds described herein may be subjected to a number of aqueous washing steps during manufacture until an isolated form is reached, where soluble compounds or residues are removed from the insoluble finished compound during manufacture.

[0035] biological polysaccharide compounds Biological polysaccharide compounds may be characterized by specific glycosyl compounds. Glycosyl chains may be the part of biological polysaccharide compounds that cause inflammatory reactions in humans. It is understood by the inventors that the described isolated biological polysaccharide compounds may have multiple glycosyl side chains, which include antigenic regions exposed within the glycosyl chains.

[0036] The biological polysaccharide compounds described above are unique in that they have a significant amount of 1:6 linked glucopyranosyl residues. Technical β-glucan compounds often have this side chain (and other side chains removed), leaving a very high proportion of 1:3 side chains. The inventors have discovered that maintaining the 1:6 side chains (and other side chains that are branched vs. unbranched) can be very important to achieving the desired efficacy.

[0037] Furthermore, the inventors have discovered that the biological polysaccharide compound provides an optimal level of immune response to achieve efficacy, without leaving any components within the yeast cells that may cause infection or undesirable effects when placed in an open wound.

[0038] Side chain analysis In at least one embodiment, the described biological polysaccharide compounds contain only the described 1:3 and 1:6 side chains.

[0039] In at least one embodiment, in addition to the 1:3 and 1:6 side chains described, additional side chains may be present. If additional side chains are present, they may include at least one of the following side chain types: 1:4 linked glucopyranosyl residues; 3:4 linked glucopyranosyl residues; 2:3 linked glucopyranosyl residues; 3:6 linked glucopyranosyl residues; 2:6 and 4:6 linked glucopyranosyl residues; 3:4:6 linked glucopyranosyl residues; Terminally bound glucopyranosyl residue.

[0040] The amount of these additional side chains can vary. In at least one embodiment, they can be present (all or some or one of) the following approximate concentrations (wt %): 2–6% 1:4-linked glucopyranosyl residues; 0.01-0.5% 3:4 linked glucopyranosyl residues; 0.5-4% 2:3 linked glucopyranosyl residues; 3:6-linked glucopyranosyl residues, 3–10%; 0.2-1% 2:6 and 4:6 linked glucopyranosyl residues; 0.01-0.5% 3:4:6 linked glucopyranosyl residues; 2–8% terminally bound glucopyranosyl residues.

[0041] In at least one embodiment, the biological polysaccharide compound may be characterized by having a glycosyl compound analysis of approximately (wt %): 67.7% 1:3 bound glucopyranosyl residues 1:6 bound glucopyranosyl residues 12.7% 1:4 bound glucopyranosyl residues 4.4% 0.3% 3:4 linked glucopyranosyl residues 2% 2:3 linked glucopyranosyl residues; 6.3% 3:6 linked glucopyranosyl residues; 0.6% 2:6 and 4:6 linked glucopyranosyl residues; 0.2% 3:4:6 linked glucopyranosyl residues; terminally bound glucopyranosyl residues, 5.8%;

[0042] Without wishing to be bound by theory, the inventors hypothesize that the presence of the 1:6 side chain in particular (and possibly other side chains of interest) may be important in achieving the strong immunogenic response observed with biological polysaccharide compounds, rather than the potentially uninhibited inflammatory cascade observed as has been noted in the art with existing biological polysaccharide compounds such as β-glucan compounds.

[0043] purity Said biological polysaccharide compounds can be characterized as being highly or ultra-pure, where purity in this context refers to the removal of cellular components such as proteins, enzymes, lipids, nuclear material from the β-glucan as well as any residues from the extraction process.

[0044] As mentioned above, the purity of the β-glucan may be 85 to 100% (by weight). The purity of the β-glucan may be 90 to 100%, or 95 to 100%.

[0045] Purity can also be measured in terms of protein or lipid impurity content.

[0046] In at least one embodiment, the biological polysaccharide compound may be characterized as having a protein content of 3, or 2, or 1, or 1, or 0.5% by weight or less. In at least one embodiment, the residual protein content in the biological polysaccharide compound may be less than 0.3%. Residual protein content may be measured by amino acid analysis.

[0047] Additionally, the biological polysaccharide compound may be characterized by having a lipid content of 3, or 2, or 1, or 0.5% by weight or less. In at least one embodiment, the residual lipid content in the biological polysaccharide compound may be less than 0.3%. The residual lipid content may be measured by gravimetric extraction.

[0048] In at least one embodiment, the biological polysaccharide compound can be characterized as being substantially free of residues from the solvents or chemicals utilized in its extraction.

[0049] The purity described may be a major advantage over the art, as it allows for consistency in dosage and results, potentially avoiding side effects and efficacy variations associated with changes in purity or presence of other compounds. Biological polysaccharide compounds, particularly β-glucan compounds, are naturally occurring compounds in nature. Isolation to high purity overcomes technical problems noted in the art, with variability in efficacy, confusing results and possible side effects or inflammatory cascades, especially for insoluble biological polysaccharide compounds as described herein.

[0050] Furthermore, to make therapeutic claims and gain regulatory approval, it is necessary to overcome the high degree of variability in the final compound that has plagued previous extractions of biological polysaccharide compounds, i.e., the prior art lacks the certainty of achieving consistency in biological polysaccharide compounds that can make consistent levels of efficacy claims and achieve drug-like properties that can achieve CMC package approval, which form key elements of regulatory drug approval.

[0051] molecular weight As noted above, biological polysaccharide compounds can be large compounds with molecular weights between 0.5 and 2.2 MDa. In at least one embodiment, the molecular weight can be between 0.5 and 2.2, or between 0.6 and 2.2, or between 0.7 and 2.2, or between 0.8 and 2.2, or between 0.9 and 2.2, or between 1.0 and 2.2, or between 1.1 and 2.2, or between 1.2 and 2.2, or between 1.3 and 2.2, or between 1.4 and 2.2, or between 1.5 and 2.2 MDa. This molecular weight can be measured as a distribution or average molecular weight.

[0052] The molecular weight can be determined by gel permeation chromatography.

[0053] Bioassay The bioassay mentioned is a measure of the inflammatory response caused by the biological polysaccharide compound, measured using a bioassay that tests the TNF-α response of the compound on human harvested macrophage cells. The biological polysaccharide compounds used do not appear to cause a runaway inflammatory cascade, but rather initiate inflammation and healing. The art shows examples where insoluble biological polysaccharides cause severe inflammatory responses that are different from those observed by the inventors with the above compounds.

[0054] sterility In at least one embodiment, the biological polysaccharide compound may be further characterized by having a very low bio-burden. In at least one embodiment, the compound may be sterile. In the context of this specification, the term "very low bio-burden" or "sterility" refers to a microbial count of less than 10 cfu / g and the absence of pathogens.

[0055] In at least one embodiment, the described compounds meet the USP51 (antimicrobial effectiveness) and USP61 (microbial limit) criteria. In at least one embodiment, the described biological polysaccharide compounds can meet the USP71 (sterility) criteria.

[0056] USP standards are standards enforced by regulatory agencies to measure sterility. One way to complete USP71 testing is to perform growth promotion tests and evaluate other quality parameters to ensure that the medium can support the growth of the six microorganisms listed in the USP71 sterility test. The medium is then inoculated using either the sealed membrane filtration method or the direct inoculation method, and the test vessel is then incubated at the appropriate temperature for at least 14 days and the microbial growth is measured. For the sample to comply with USP71, there should be no evidence of growth at the end of the incubation period and the drug product is given a "sterile" result indicating that no contaminating microorganisms were found in the sample examined under the test conditions.

[0057] To further illustrate this, the described biological polysaccharide compounds can be further characterized by having the following sterility criteria: TIFF2024528128000002.tif41170

[0058] fine particles The biological polysaccharide compound may be in the form of a particulate. The particle size may be less than 40 μm. This may be measured using the Malvern particle size test method.

[0059] Presentation of isolated biological polysaccharide compounds as microparticles can be useful to increase the surface area and expose more of the antigenic regions of the biological polysaccharide compounds (mainly the 1-3 and 1-6 glycosyl side chains).

[0060] exterior The isolated biological polysaccharide compound may be white to slightly off-white in color and may be a powder in its pure form.

[0061] Residual Solvent Technical methods of isolating β-glucan compounds / biological polysaccharide compounds may rely on various solvents / reagents including but not limited to water, chloroform, ethanol, etc.

[0062] In the inventors' experience, virtually no residual solvent is detectable in the isolated biological polysaccharide compounds described herein.

[0063] stability The biological polysaccharide compounds are highly stable. In tests completed by the inventors, the isolated biological polysaccharide compounds are shelf stable for at least 1, or 2, or 3, or 4, or 5 years after storage at ambient temperature. The biological polysaccharide compounds also appear to be heat stable. In one trial completed on behalf of the inventors, a thermogravimetric analysis was completed that demonstrated that the compounds remained stable even after treatment at 220° C., a very high temperature given stability.

[0064] This may be a result of the high purity and sterility of the isolated biological polysaccharide compounds and therefore may be the reason why these compounds show little degradation even when stored for long periods of time.

[0065] Treatment method In a second aspect, there is provided a method of treating the skin of a patient in need thereof by topical application to a wound site of a vehicle, the vehicle comprising a therapeutically effective amount of an isolated biological polysaccharide compound substantially as described herein.

[0066] Further examples of specific methods are described further below.

[0067] use In a third aspect, there is provided the use of an isolated biological polysaccharide compound, substantially as described herein, in the manufacture of a medicament for the topical treatment of the skin of a patient in need thereof.

[0068] Further examples of specific uses are described further below.

[0069] Effectiveness The inventors have discovered that the described biological polysaccharide compounds may be optimal immunogenic molecules that can be utilized in wound healing.

[0070] A summary of the efficacy demonstrated from two recent Phase IIB double-blind randomized clinical trials completed in 2020 is provided below and further illustrated in the Examples and Figures.

[0071] chronic wound In a randomized, double-blind, placebo-controlled Phase IIB trial of 82 patients, the biological polysaccharide compound showed a strong signal of efficacy compared to a vehicle gel containing: The adjusted difference in incidence of complete closure in the per-protocol group vs. placebo was 27% (p=0.1). The adjusted difference in incidence of complete closure in the intention-to-treat group vs. placebo was 22% (p=0.12). It should be noted that a 10% difference in the incidence of complete closure is considered clinically meaningful in the art.

[0072] Cosmetic procedures A Phase IIB, double-blind, placebo-controlled trial of 40 patients evaluated the efficacy of the described biological polysaccharide compound by measuring skin quality and healing after fractionated laser treatment. The biological polysaccharide compound demonstrated the following: - 70% reduction in wrinkles compared to the placebo group (P<0.04). 114% improvement in elastosis (P<0.13), doubling the improvement in elastosis by 28 days.

[0073] The mechanism of immune stimulatory action can be tailored to suit different indications, allowing for multiple applications.

[0074] It is the inventors' understanding that the identified isolated biological polysaccharide compounds stimulate the innate dermal immune system by mimicking a biological threat (e.g., a yeast infection), which causes the immune system to respond via activation of the NF-κB pathway in macrophages.

[0075] Macrophages regulate and control wound healing responses. The described biological polysaccharide compounds stimulate a simple antigen response mechanism that induces and accelerates the natural cascade of wound healing responses, but does not cause an uncontrolled inflammatory response. The result appears to be an unexpectedly high efficacy in regenerating new tissue and closing the wound.

[0076] The isolated biological polysaccharide compounds are believed to activate pattern recognition receptors on immune cells that developed during human evolution to defend against pathogenic microorganisms.

[0077] Antigenic regions of biological polysaccharide compounds, such as yeast glucan, may be recognized by the body's macrophages as decoys to a perceived threat, triggering stimulation of the toll-like receptor (TLR) 2 and dectin-1 membrane receptors on wound macrophages and signaling pathways aimed at combating the threat. However, while the decoys do not cause the perceived damage, the resulting macrophage activation is thought to stimulate wound healing in a variety of beneficial ways.

[0078] The inventors understand that the method stimulates wound macrophage activity, resulting in increased phagocytosis, increased secretion of wound healing cytokines, and stimulation of angiogenesis and wound repair.

[0079] In the inventor's experience, no side effects have been observed and no significant inflammation has been measured with this method / use, which is completely contrary to what the art suggests for insoluble biological polysaccharide compounds.

[0080] Vehicle In one embodiment, the vehicle may be a gel composition comprising the isolated biological polysaccharide compound. The vehicle may be a high viscosity gel. For example, the viscosity of the gel may be greater than 3000 cps. The gel may be free of chemical preservatives, such as paraffin or traditional chemical preservatives. This may be desirable to avoid interference or adverse effects on an open wound. The gel may be an aqueous gel. The biological polysaccharide compound may be in the form of a particulate. As mentioned above, the biological polysaccharide compound is insoluble, so the biological polysaccharide compound may be suspended in the gel as a particulate suspension.

[0081] The vehicle can also take other pharma- ceutically and physiologically acceptable forms. The vehicle can generally be aqueous. The vehicle can act to suspend or hold the biological polysaccharide compound as a particulate suspension or particles on the substrate until topically applied. Examples of alternative vehicles include creams, ointments, bandages, medical devices used in the treatment of the skin, such as devices for treating wounds and burns, and the like.

[0082] Concentration / dosage of biological polysaccharide compounds In at least one embodiment, the vehicle may comprise 0.05, or 0.06, or 0.07, or 0.08, or 0.09, or 0.1, or 0.2, or 0.3, or 0.4, or 0.5, or 0.6, or 0.7, or 0.8, or 0.9, or 1.0, or 1.1, or 1.2, or 1.3, or 1.4, or 1.5% by weight of the isolated biological polysaccharide compound. In at least one embodiment, the vehicle comprises 0.05-1.5% or 0.1-1.0% by weight of the isolated biological polysaccharide compound.

[0083] The vehicle may be applied topically to the wound site daily or twice daily. The vehicle may be applied for multiple days or for several weeks. The dosing regimen may be a function of the concentration of the compound in the vehicle, the treatment being administered, and the response of the patient, among other factors. In the inventors' experience, treatment regimens tend to be one to two applications per day for 5-14 days, or, in the case of treatment of chronic wounds, one, two, or three times per week for a treatment period of 12-20 weeks. This period may vary.

[0084] The vehicle can be applied by the patient to their own skin site at home.

[0085] The vehicle containing the compound may be applied to the site as a layer 1-5 mm thick, which may be absorbed into the skin over time.

[0086] Treating chronic wounds The method / use can be used to treat chronic wounds. The chronic wound can be an ulcer. The ulcer can be a venous leg ulcer. According to the inventor's experience, the biological polysaccharide compound has an unexpected synergistic effect in the treatment of venous leg ulcers compared to placebo.

[0087] In at least one embodiment, the inventors report that the described biological polysaccharide compounds, when used to treat venous leg ulcers in a Phase IIB double-blind, randomized, placebo-controlled clinical trial of 67 patients, reduced ulcer size by 2 to 12 cm. 2 They found that for chronic venous leg ulcers, there was an approximately 27% difference in the incidence of complete wound closure compared to vehicle gel. This level of efficacy is likely superior to currently approved competing products.

[0088] The authors also found that the reduction in wound area in chronic venous leg ulcers was twice as high with the biological polysaccharide compound (91%) compared to 46.6% with placebo. These findings are further illustrated in the Examples below.

[0089] Cosmetic procedures The method / use may be a cosmetic skin treatment. In at least one embodiment, the effectiveness of the cosmetic skin treatment may be measured in terms of cosmetic skin quality, in particular by skin elastosis and wrinkles. According to the inventors' experience, the described biological polysaccharide compound has unexpectedly produced synergistic effects in cosmetic skin treatment.

[0090] In at least one embodiment, use of the gel following a controlled burn procedure, a CO2 fractionated laser treatment of the chest, in a Phase IIB, double-blind, placebo-controlled, 42-patient trial is described, in which the described biological polysaccharide compound resulted in nearly doubling the improvement in skin quality following laser ablation, with elastosis 75% vs. 35% for placebo (p<0.01) and wrinkle percentage 85% vs. 50% for placebo (p<0.04).

[0091] Further or Related Effects The biological polysaccharide compounds described above may have additional tissue repair and healing effects, including one or more of the following: Optimizing skin healing and skin quality; Reducing skin wrinkles after cosmetic procedures; Increasing skin elastosis during healing; Treatment of post-fractional, fully ablative, post-resurfacing skin (a known treatment option to improve the appearance of UV photodamage, cheilitis, pigmentation, and suboptimal skin texture); A supplementary post-operative topically applied gel to promote skin texture benefits.

[0092] The biological polysaccharide compound, when in gel form as described above, can be applied in any cosmetic treatment involving any wound healing, including healing after one or more of the following treatments / procedures: incision; puncture; Ablation; Severe chemical peels; skin graft; Invasive and mild laser procedures.

[0093] Application of gels containing biological polysaccharide compounds may enhance the clinical effectiveness of cosmetic procedures resulting in improved skin quality and healing. This is believed to be because the described biological polysaccharide compounds stimulate the body's own processes for tissue regeneration and collagen production in response to damage caused by the underlying cosmetic procedure, be it laser treatment, chemical peel, incision, or other forms of ablation.

[0094] Method for producing biological polysaccharide compounds In a fourth aspect, Selecting the yeast cells; lysing the cells and collecting cell wall fragments; heating and acidifying the cell wall fragments to remove mannan and chitin; performing a phase separation using a solvent to remove additional mannan and additional chitin along with proteins, glycogen and lipids; separating the solvent and other non-polysaccharide compounds by boiling and drying; performing at least one water rinsing step after dissolution and before acidification; There is provided a method for producing an isolated biological polysaccharide compound substantially as described above, comprising:

[0095] Dissolution In at least one embodiment, lysis can occur by alkaline or heat treatment, or both. Lysis is a common technique, although other methods such as pressure fluctuations, sonic disruption, homogenizers, enzymes, detergents, etc. can also be used.

[0096] Water Rinse The method may further comprise at least one water rinse after said acidification and before said phase separation. Additional water rinses are contemplated to complete additional purification, but are performed in a gentle manner that preserves the glycosyl branching structure of the polysaccharide, as opposed to more harsh technical methods that may cause alterations in the polysaccharide structure.

[0097] pH change In addition to rinsing with water, after acidification and prior to the phase separation, the collected mixture may be subjected to a pH change and / or alcohol wash. In at least one embodiment, the pH may be lowered to 4.0 to wash the alcohol, then raised to 9.0 to wash the alcohol, and then adjusted to 7.0 to wash the alcohol. Alcohol and water rinses may also be performed after phase separation. The alcohol used may be selected from one or more lower alcohols. Examples of alcohols include methanol, ethanol, propanol, and combinations thereof.

[0098] solvent The solvent used above may be an organic solvent. The solvent used may be a non-polar solvent. The specific gravity of the solvent may be 1.0 or more. Examples of solvents that may be used include one or more of the following: methylchloroform, chloroform, dichloromethane, tetrachloroethane, carbon tetrachloride, ethyl acetate, and combinations thereof. This solvent extraction process may be completed at room temperature. It is understood that the use of this type of solvent may remove lipids from the mixture and thus aid in the purification of biological polysaccharide compounds.

[0099] Phase separation using said solvents can be completed at room temperature and neutral pH. Based on the inventors' experience, no special conditions are required.

[0100] Boiling and drying In at least one embodiment, the method can further include, after separation of the solvent and other non-polysaccharide compounds by boiling and drying, further purification through at least one additional series of solvent rinses, alcohol rinses, and optionally additional water rinses before drying the final product again. The solvents used in these additional steps can be the same as those used above for phase separation. The alcohols used in these additional steps can be the same as those used above for the alcohol washes. The water rinses in this additional step can be performed using hot water (>50°C). The drying described can be performed in one step or in multiple steps using multiple drying techniques, such as spray drying, oven drying, freeze drying, vacuum drying, etc.

[0101] It should be understood that the above process is quite different from the closest known technology, such as the process described in US 6,242,594, in which the process described is a four-step process, whereas the above process requires numerous additional steps, including extensively various washing and solvent steps, as well as additional purification and drying steps.

[0102] For pilot or commercial scale production of polysaccharides, the inventors added additional water rinsing steps to stop the accumulation of large amounts of salt crystals. These water rinsing steps may have the added benefit of removing non-polysaccharide material and providing purification at an earlier stage in the process. The water rinsing process may also be important to separate branched and non-branched molecules and collect a higher percentage of the branched 1-3:1-6 compounds in the final isolated product.

[0103] Different Structures As noted above, the methods described produce compounds with structures (and potencies) that are fundamentally different from those described in the prior art. For example, the methods described herein result in isolated biological polysaccharide compounds having: Substantially higher molecular weight than the prior art (molecular weight of 60k-250k Daltons (average 140k Daltons) versus the order of 0.7m-2.2m Daltons) as described in US 6,242,594. · Larger, more complex molecules consisting of a greater proportion of branched 1-6 side chains. US 6,242,594 teaches the preparation of a compound containing 96%-97% 1-3 chains and 3-4% 1-6 chains. The compounds isolated from this method and described herein have a much higher proportion of branched 1-3 and 1-6 linkages compared to unbranched 1-3 and 1-6 side chains. The isolated compounds produced by the methods described herein produce ultra-pure polysaccharides with less than 2% protein or lipid, which is generally much higher than that described in the art, including U.S. Pat. No. 6,242,594. A compound with a very low bioburden, with minimal, if any, extraneous cellular material present in the final compound. This can be very important given that one of the applications of the isolated compound may be for use in open wounds.

[0104] The isolated biological polysaccharide compounds, uses and methods of production described above, as demonstrated above, may include many advantages. Some examples of the advantages include one or more of the following: Providing optimal immunogenic compounds designed for wound healing with optimal levels of immune response without adverse inflammatory effects. Clinically validated to improve healing of chronic wounds (double-blind, randomized, placebo-controlled trial superior to gold standard therapy. Nearly 2-fold improvement in skin quality after standard laser treatment in a double-blind, placebo-controlled human study. The manufacturing method appears to solve existing manufacturing challenges with its ability to produce well-characterized molecules that deliver measurably consistent potency levels batch-to-batch. · Signs of reduced itching and pressure after treatment.

[0105] The above-described embodiments may be broadly described as consisting of the parts, elements, and features referred to or indicated in the specification of this application, individually or collectively, and any or all combinations of any two or more of said parts, elements, or features.

[0106] Furthermore, where specific integers that have known equivalents in the art to which the embodiments pertain are referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0107] Working example Next, the above polysaccharide compounds, their use and production methods will be described with specific examples.

[0108] Example 1 Table 1 below provides a complete characterization of the isolated biological polysaccharide compounds described above.

[0109] TIFF2024528128000003.tif249170TIFF2024528128000004.tif78170

[0110] Example 2 In this example, methods and results of glycosyl linkage analysis are described that are used to assist in the characterization of the isolated biological polysaccharide compounds described herein.

[0111] method For glycosyl linkage analysis, samples (1.2 mg) were first dissolved in 400 μL of DMSO. The samples were then permethylated, depolymerized, reduced, and acetylated. The resulting partially methylated alditol acetates (PMAA) were analyzed by gas chromatography-mass spectrometry (GC-MS) as described by Heiss et al. (2009) Carbohydr. Res. 344:915.

[0112] Permethylation was carried out by two treatments of 400 μL sodium hydroxide (15 min) and 100 μL methyl iodide (45 min). The reaction was quenched by adding 2 mL water and excess methyl iodide was removed by nitrogen flow. After extraction with dichloromethane, water washing (3 times) and removal of the solvent, the permethylated material was hydrolyzed using 2M TFA (400 μL, 2 h at 121 °C in a sealed tube), reduced with NaBD4 and acetylated using acetic anhydride / TFA (250 μL + 230 μL, 15 min, 50 °C). The resulting PMAA was analyzed on an Agilent 7890A GC connected to a 5975C MSD (mass selective detector, electron impact ionization mode). Separation was carried out on a 30 m Supelco SP-2331 bonded-phase fused silica capillary column.

[0113] result Linkage analysis showed that the most abundant glycosidic linkages of glucose were 3-linked glucopyranosyl residues, which were larger than 6-linked glucopyranosyl residues. The ratio of 6-Glc to 3,6-Glc was approximately 2 to 1, suggesting an average side chain length of 3 Glc residues, and the ratio of 3-Glc to 3,6-Glc was approximately 11 to 1, suggesting an average side chain on every 10th backbone Glc residue. Table 2 below shows the identified linkages and their relative abundance.

[0114] TIFF2024528128000005.tif69170

[0115] Example 3 This example describes the methods and results of NMR studies used to assist in the characterization of the isolated biological polysaccharide compounds described herein.

[0116] 1H NMR samples were prepared by dissolving samples of the compounds of the invention in D6MSO and D-TFA. The samples were tested using a 400 MHz NMR instrument. The test results are shown in Table 3 below and in Figure 1A. The anomeric H1 and H6 proton signals of the main chain and side chains were identified. The ratio of 1-3-Glc main chain to 1-6-Glc branches was calculated based on the NMR data. On average, the side chains were about 4 Glc residues in length. The ratio of 1-3-Glc to 1-6-Glc was about 10 to 1, suggesting that a side chain is attached to every 10th backbone Glc residue on average.

[0117] Table 3 below shows the NMR summary output.

[0118] TIFF2024528128000006.tif34170

[0119] The molecule is then represented diagrammatically as shown in Figure 1B, where the biological polysaccharide of the present invention is a β-glucan-based sugar consisting of 3- and 3,6-linked glucose residue units. Figure 1B shows the general structure of a (1->3)(1->6)-β-glucan. For simplicity, the key residues are labeled "BC", "Br", and "SC". BC represents the main chain residues, Br represents the branching residues, and SC represents the side chain residues.

[0120] Example 4 In this example, an overview of the clinical trials that the applicant has completed to date is provided.

[0121] Over 200 patients have been studied across Phase I, IIA and IIB clinical trials with the compounds of the invention, most of which (over 150 subjects) were conducted in double-blind, randomized, placebo-controlled studies, with the majority of patients based in the United States, with the remainder in studies conducted in Australia.

[0122] Recently, the applicant completed a Phase IIB clinical trial program across two indications: the treatment of chronic wounds and cosmetic dermatology.

[0123] A randomized, double-blind, placebo-controlled Phase IIB chronic wound trial was completed in 2020 under an FDA-approved protocol, and the results showed efficacy comparable to, if not superior to, the gold standard therapy.

[0124] A Phase IIB randomized, double-blind, placebo-controlled clinical trial in cosmetic dermatology following fractionated CO2 laser irradiation of the chest demonstrated nearly twice the improvement in skin quality compared to placebo gel at 28 days after treatment.

[0125] All Phase II trials were conducted recruiting randomized trial subjects using FDA-approved protocols who were assigned to treatment or control groups using a chance element to reduce selection and / or allocation bias.

[0126] Double-blind: Neither the participants nor the administerers know whether they are receiving a placebo or the study treatment.

[0127] Controlled placebo: The control group received an ineffective "placebo" treatment that was specifically designed to have no real effect, in order to again benchmark the treatment being studied.

[0128] Table 4 below provides an overview of the clinical validation to date.

[0129] TIFF2024528128000007.tif140170

[0130] Example 5 A Phase I trial was conducted in Australia to establish the safety and efficacy of the isolated biological polysaccharide compound described above. An open, uncontrolled study was conducted in six patients. The compound was applied topically every 2-3 days for four weeks to patients where standard wound treatments had failed.

[0131] Analysis and determination of wound area reduction by planimetry was evaluated 56 days after the start of treatment and compared with pre-treatment wound area. In addition, safety and toxicity analyses were also performed.

[0132] No significant intolerance or toxicity was observed or reported in relation to the use of the test product. Healing responses were observed in all six patients, with reductions in wound surface area ranging from 26% to 82% measured over the 56-day period.

[0133] As shown in FIG. 2, an average 56% reduction in wound size in wound surface area was observed on day 56 compared to day 1 of treatment.

[0134] In summary, this Phase I study confirmed the ability of compounds of the invention to stimulate healing in chronic trophic ulcers.

[0135] Example 6 In this example, a Phase I / II clinical trial is described.

[0136] A second Phase I / II study was conducted to determine the efficacy of the compounds of the present invention, which were studied in a single-center, randomized, double-blind, vehicle-controlled study in 18 patients with CDVI ulcers that had become refractory to standard wound management therapy. The compounds of the present invention were compared to another form of glucan (Glucodine™) with a lower molecular weight range and a lower percentage of (1-6)-β-glucan side branches, and to vehicle (control).

[0137] Two glucan materials (invention and Glucodine™) were suspended in a cream base (0.1% w / w; paraben preserved).

[0138] Patients were randomly assigned to three treatment groups (two active groups and one vehicle control group), with six patients per group.

[0139] Treatment was three times a week for four weeks.

[0140] No intolerance or toxicity was observed or reported in association with the use of either the active test article or the vehicle control.

[0141] Efficacy was assessed by planimetry, measuring the surface area of ​​the wound. The primary efficacy parameter was improvement in ulcer surface area from baseline (Visit 1) to the end of the study (Visit 6). The mean improvement over 4 weeks was 4.4% in the vehicle group, 36.7% in the compound of the invention group, and 17.3% in the Glucodine™ group. The extent of healing over 8 weeks is as shown in Table 5 below and in Figure 3 (note that the compound of the invention is labeled "Glucoprime" in Figure 3).

[0142] TIFF2024528128000008.tif26170

[0143] The results showed that the compounds of the present invention are highly effective as wound healing agents.

[0144] Example 7 This example describes a Phase 2A clinical trial of the use of compounds of the invention to treat chronic wounds, in this case venous ulcers.

[0145] The change in formulation was driven by a strategic decision to avoid preservatives such as parabens (used in the second clinical study) that may have an inhibitory effect on macrophages. The decision was made to revert to the methanol preservative used in the first clinical study. Formulation studies were then conducted, leading to the decision to use a gel base preserved with varying degrees of preservative.

[0146] Formulations containing 0.1% and 1.0% active substance were tested.

[0147] This was a phase 2, double-blind, randomized, vehicle-controlled study in which patients were assigned to one of three treatment groups on a randomized basis using a computer-generated allocation sequence. Fifty-eight patients (36 males, 22 females) with chronic venous ulcers were recruited at two centers and randomly assigned to either high-dose active (1.0% compound of the invention gel) or low-dose active (0.1% compound of the invention gel), or gel base alone (vehicle control). Patient ages ranged between 34 and 93 years, with comparable demographic characteristics between the three treatment groups. The treatment was applied over the entire wound surface to a depth of approximately 3 mm, three times a week for 12 weeks, during standard wound care management. This investigation aimed to provide a statistical evaluation of the efficacy and safety of the compound of the invention in patients with chronic venous insufficiency ulcers of the lower extremities.

[0148] As part of the study, the ulcers were cleaned and debrided, the test product was applied, and the wounds were covered with a pressure dressing three times a week for up to 12 weeks. Wound margins were tracked weekly to measure both the rate and extent of healing.

[0149] No drug-related toxicity occurred during the study in either active group.

[0150] Data from a Phase II study showed that the compounds of the present invention accelerate the rate of wound healing, with ulcers treated with the compounds of the present invention healing at a statistically significant improvement rate compared to ulcers treated with placebo. The wound closure rates obtained in this study are shown in Table 6 below.

[0151] TIFF2024528128000009.tif44170

[0152] The overall mean levels of healing over 12 weeks, as measured by reduction in ulcer surface area, were as follows in Table 7:

[0153] TIFF2024528128000010.tif40170

[0154] The median percent change from baseline in ulcer area is also shown in FIG. 4, where "High (1.0%)" refers to the high dose samples and "Low (0.1%)" refers to the low dose samples.

[0155] These results were observed despite large differences in ulcer size, with the two compound of the invention treatment groups having substantially larger mean ulcer sizes than the placebo group, regardless of patient randomization.

[0156] Example 8 A summary of the complete results of the Phase IIA trial is shown in Table 8 below.

[0157] Table 8 - Results of the Phase 2A Australian Chronic Venous Ulcer Trial TIFF2024528128000011.tif191170

[0158] As shown in the results above, the compounds of the present invention accelerated the rate of wound healing. With the compounds of the present invention, the treated ulcers healed at a significantly faster rate (mm per day) compared to the placebo-treated ulcers. 2 ) was cured. The overall mean cure levels over 12 weeks were 10% (placebo), 59% (low dose of compound of the invention), and 55% (high dose of compound of the invention).

[0159] Example 9 This example describes a Phase IIA burn trial. The US study was completed as a double-blind, randomized, placebo-controlled Phase 2 study investigating the safety and efficacy of topically applied 0.1% and 1.0% invention compound (vehicle was gel) compared to placebo in patients undergoing carbon dioxide laser skin resurfacing (LSR) of the lower eyelid.

[0160] Design and Endpoints: This study evaluated the safety and efficacy of two concentrations of the invention compound gel (referred to as GLYC-101 at 0.1% concentration and GLYC-101 at 1.0% concentration) in promoting wound healing in the skin of the lower eyelids of 26 subjects undergoing CO2LSR for cosmetic purposes (wrinkle reduction) compared to placebo (gel-based). The study compound was applied topically to the excised skin area of ​​each lower eyelid such that the entire wound surface was covered with a layer of test article (either placebo or GLYC-101) approximately 0.5 mm (not to exceed 1 mm) thick. Test material was applied daily (approximately 24 hours after the previous application) for a total of 5 days. Overall, treatment with the invention compound (GLYC-101 1.0% or GLYC-101 0.1%) was safe and well tolerated when compared to placebo.

[0161] RESULTS:The investigational drug did not appear to increase the incidence or exacerbate existing safety issues associated with postoperative CO2LSR ablation of the lower eyelid area for wrinkle removal, nor did it appear to reduce the number or severity of safety events.

[0162] Comparison of each GLYC-101 (the compound of the present invention is now named TR-987) group with placebo for the primary endpoint (time to complete wound closure) showed positive results when considering the complete subject data set from all treatment combinations. Figure 5 shows the Phase 2 study laser data time to wound closure. There was an average 30% improvement in wound healing time. More specifically, when the compound of the present invention (GLYC-101 0.1% and GLYC-101 1.0%) groups were compared with the placebo group, the efficacy results of time to complete wound closure were more rapid (p=0.0062 and 0.0331, respectively). These results demonstrate the efficacy of the compound of the present invention.

[0163] Example 10 In this example, with reference to Figures 6, 7 and 8, the results of a Phase IIA clinical trial are shown.

[0164] In particular, Figures 6, 7 and 8 show an atypical example of wound size reduction in venous ulcer wounds in a completed study using a compound of the invention. Across the 58 patients in the completer cohort, a statistically significant 45% reduction was demonstrated over 12 weeks for those receiving the compound of the invention (p<0.008).

[0165] Examples of patients receiving treatment with the compounds of the invention are shown based on the Australian Venous Ulcer Trial. As shown in the images, ulcer formation is significantly reduced over time, with healing or near complete healing occurring within 8-12 weeks in the three examples shown.

[0166] Example 11 In this example, we describe a study showing wound closure findings from a Phase IIb clinical trial.

[0167] A double-blind, placebo-controlled, randomized Phase IIB clinical trial evaluating a compound of the invention versus a placebo gel in the treatment of chronic venous ulcers. A Phase IIB study has been completed.

[0168] Overall, 82 subjects were randomized to treatment (42 compounds of the invention, 38 placebo) and underwent at least one post-baseline assessment of ulcer area (ITT population). The per-protocol population was defined by the sponsor. The PP population excluded subjects who withdrew early, had major protocol deviations, or had consecutive infections.

[0169] The main criteria for the clinical trial include: · Phase IIB venous leg ulcer trial. The trial was ambitious and powerful. Double-blind, placebo-controlled. · FDA approved protocol based on valid IND. VLUs treated with an inventive compound plus standard of care, compression bandage vs. placebo gel and the same standard of care. The placebo gel was a true vehicle control that could be disguised from the active arm in every respect. - Adopted at 10 locations in the US and 3 locations in Australia. -Treatment duration is 12 weeks. 2~20cm 2 Treat ulcers 2-12 cm 2 Protocol variations to test for ulcers. · To ensure that only poorly healing ulcers were included in the study, a run-in period was made part of the inclusion criteria so that fast-healing patients who showed more than a 30% reduction were excluded from randomization. There was no age limit for ulcers. Some of the ulcers enrolled were still present approximately 300 weeks before randomization.

[0170] The analysis showed that while the primary endpoint of time to healing did not differ between groups, the key secondary endpoint of percentage of wounds that achieved 100% closure showed a strong signal of efficacy. The percentage of wounds that achieved 100% closure is considered the FDA's gold standard endpoint for healing.

[0171] In summary, a critical initial analysis of the incidence of wound closure and wound area reduction between active and placebo showed that the trial: 2~12cm 2 In the ITT group with ulcers (n=67), there was an adjusted difference in the incidence of complete closure of 20.6% (p=0.12). 2-12cm for each protocol group (n-69) 2 For ulcers with bronchospasm, there was a 27% difference in the incidence of complete closure (p=0.1). -Double the reduction in wound area in chronic venous leg ulcers (VLUs): 22-12cm per protocol group (p=0.035) 2 For ulcers, 91% compound of the invention vs. 46.6% placebo.

[0172] The additional objective of reduction in wound area also showed a strong signal of effect, and overall pain reduction compared to placebo (which was statistically significant) showed an even stronger signal of effect.

[0173] Figure 9 shows the difference between the raw incidence of complete closure between vehicle and active groups for compounds of the invention and Phase III data from the Apiligraf™ Phase III label. Apiligraf™ is perhaps the gold standard for VLU closure, reimbursing approximately $1200 per application, and routine treatment of a VLU may require 3-15 applications.

[0174] The compound of the invention per protocol group from the Phase IIB clinical trial are patients who completed the study and received the full dose of the drug for 12 weeks (the inventors believe this group to be an accurate measure of the efficacy of the drug).

[0175] The ITT group included all randomized patients, including all premature discontinuations who did not receive the full 12 weeks of treatment.

[0176] A clinically meaningful difference is generally considered to be a difference of +10% in absolute closure.

[0177] As shown in Figure 9, the compound of the present invention achieves an adjusted difference in complete closure incidence of 20.6% compared to Apiligraf™'s complete closure incidence of 17%. A meaningful difference between the ITT and PP groups is noted.

[0178] Adjusted data are based on logistic regression (compounds of the invention) and Cox regression (Apiligraf™) controlling for factors known to affect healing between groups (e.g., baseline, ulcer size, etc.).

[0179] Given the differences in inclusion and exclusion criteria of the trial designs (including blinding), the results are not strictly comparable but provide a signal of equivalent or superior efficacy for the compounds of the invention.

[0180] FIG. 10 shows the recorded logistic regression odds ratios ("OR") of 100% cure achievement for the active treatment group versus placebo. Output is from a logistic regression adjusted for covariates known to affect cure. Interpretation of the OR, for example, if the OR is 1.4, the odds of cure for the active treatment group are 1.4 times higher than the odds of cure for the placebo group. Values ​​greater than 1 favor treatment and values ​​less than 1 favor placebo.

[0181] All recorded ORs were >2 in favor of treatment, suggesting that the odds of cure in the active group were more than twice as high as the odds of achieving complete cure in the placebo group. Figure 10 shows protocol 2-20 cm 2 and 2 to 12 cm 2 Figure 1 shows the ORs for complete cure for both size groups identified in ITT and PP groups, respectively. The upper and lower confidence intervals are also shown.

[0182] Since odds ratios for all groups are clinically significant and all groups favor treatment, it is hypothesized that as "n" increases (in a Phase III trial), the presented confidence intervals will narrow and statistical significance will be achieved. The results show that patients were more than twice as likely to achieve 100% closure in the compound of the invention group than in the vehicle or placebo groups.

[0183] Example 12 In this example, the reduction in mean wound area in Phase IIA and Phase IIB clinical trials is described with reference to Figure 11. This data demonstrates that compounds of the invention produce consistent efficacy as measured by reduction in wound area in two independent Phase II clinical trials.

[0184] The mean reduction in wound area is considered by the FDA as an acceptable endpoint for Phase II clinical trials to measure efficacy in terms of healing. It is only accepted by the FDA as a valid Phase II endpoint because it is considered a precursor or signal of healing. It is not accepted as a valid Phase III marker. The results of the compounds of the invention by protocol group from the Phase IIB clinical trial show the results of patients who completed the study and received all the drug, and the completer cohort of the Phase IIA clinical trial, which are patients who completed the study.

[0185] FIG. 13 shows a comparison of the effectiveness of the present invention using the metric of reduction in wound area compared to Epifix™.

[0186] The Epifix™ data used was from a published sponsor-funded clinical trial (not through the FDA) that was not double-blind, i.e., the physicians were not blinded. Epifix™ is a biological patch similar to Apiligraf™ that has been approved as a human tissue product and has achieved reasonable commercialization. Epifix™ reimburses over $1000 per application, but requires multiple applications to close. More information about Epifix™ is described at https: / / mimedx.com / epifix / .

[0187] As shown in FIG. 13, the compound of the present invention achieved a nearly two-fold difference in mean wound area reduction of 91% vs. 47% with placebo (labeled TR-987) and an absolute difference of 44% with Epifix™ (p=0.035), achieving an absolute difference of 25% between the compound of the present invention and placebo (p<0.02).

[0188] Example 13 In this example, the compounds of the invention were compared to the technology product Apiligraf™ (see www.apiligraf.com) for venous leg ulcer treatment (VLU). Figure 12 shows the difference between the raw incidence of complete closure between the vehicle and active groups for the compounds of the invention and the Phase III data from the Apiligraf™ Phase III label. Apiligraf™ is likely the gold standard for VLU closure, reimbursing approximately US$1200 per application and may require 3-15 applications.

[0189] The compound of the invention per protocol group from the Phase IIB trial are patients who completed the trial and received the full dose of drug for 12 weeks (this group is an accurate measure of the efficacy of the compound).

[0190] The ITT group included all randomized patients, including all withdrawals.

[0191] A clinically meaningful difference is generally considered to be a +10% difference in 100% wound closure.

[0192] The compound of the invention achieved an adjusted difference versus placebo of 21% in the incidence of complete closure, whereas the adjusted incidence of complete closure for Apiligraf™ was 17% versus placebo, demonstrating superior differentiation of the compound of the invention versus the presumably gold standard product in both the ITT and PP groups.

[0193] The 21% adjusted difference for the study of the compound of the present invention (designated TR-987) is the absolute difference in complete cure observed between TR-987 and the placebo used in that study.

[0194] Adjusted data are based on logistic regression (compounds of the invention) and Cox regression (Apiligraf™) controlling for factors known to affect healing between groups (e.g. baseline ulcer size).

[0195] Given the differences in inclusion and exclusion criteria in study designs (including blinding), the results are not strictly comparable, but provide a strong signal of comparable efficacy for the compounds of the invention.

[0196] Example 14 In this example, a test is shown comparing the compound of the present invention with the conventional product Epifix (trademark) to compare the reduction in wound area. The reduction rate of wound area between the compound of the present invention (labeled TR-987) and Epifix (trademark) is shown in Figure 13.

[0197] Mean wound area reduction is considered an acceptable endpoint by the FDA only for Phase II trials and for purposes of measuring efficacy, not for Phase III or pivotal trials.

[0198] Compounds of the invention are present in per protocol groups from a Phase IIB trial that are patients who completed the trial and received the full dose of the drug (this group is an accurate measure of the efficacy of the drug).

[0199] The Epifix™ data is from a sponsor-sponsored clinical trial (not through the FDA) that is not double-blind, i.e., a physician-administered comparison of a new active ingredient with a treatment.

[0200] The compound of the invention achieves nearly double the difference in mean wound area reduction, 91% vs. 47% with placebo, and compares with Epifix™ achieving an absolute difference of 44% (p=0.035) vs. 25% (p<0.02).

[0201] The reduction in wound area with the compound of the invention mirrors the earlier Phase IIA trial data: 55% vs. 25% (p<0.01).

[0202] Epifix™ is a biological patch similar to Apiligraf™ that has been approved as a human tissue product and has achieved significant market penetration. Epifix™ is reimbursed at c+$1000 per application, but requires multiple applications to close.

[0203] Given the differences in study designs, inclusion and exclusion criteria, and levels of patient and physician blinding, the results are not strictly comparable, but provide a strong signal of comparable efficacy for the compounds of the invention.

[0204] Example 15 In this example, a Phase IIB venous leg ulcer trial, n=67 (2-12 cm 2 To show a typical example of a VLU wound healed with a compound of the invention in a 2000 mouse model, the results of a Phase IIB clinical trial are illustrated with reference to Figures 14 and 15.

[0205] Figures 14 and 15 show that the ulcer size at randomization was 7.53 cm 2Another example from the study shows that ulcers were healed in 12 weeks when ulcers were present at The Heidelberg Repatriation Hospital, Melbourne, Australia. The ulcers had been open for more than 4 years prior to enrollment. The ulcers closed after 10 weeks of treatment with TR-987.

[0206] Example 16 The compounds of the present invention have been identified by applicants as useful post-treatment topical gels that improve skin quality and nearly double the clinical efficacy of cosmetic treatments in 28 days.

[0207] Figure 17 shows an atypical example of a patient administered a compound of the present invention after a minimally invasive cosmetic procedure (CO2 fraction laser treatment of the breast) and the atypical skin quality improvement results found in a Phase IIB laser ablation study n=42. The image on the left shows the laser treatment after breast fraction. The image on the right shows the same breast 28 days after treatment and after daily treatment of the treated skin using a compound of the present invention.

[0208] Patients using the compounds of the invention in a Phase IIB study nearly doubled the incidence of accelerated skin quality as measured by elastosis and wrinkling at 28 days from use of the gel vs. placebo gel + standard of care (p<0.04, n=40).

[0209] Ablative fractional lasers have been increasingly utilized in recent years to improve the appearance of UV photodamage, skin wrinkles (scars), and scars. Postoperative skin care is critical to promote optimal wound healing after treatment, but there are currently no gold standard postoperative medications.

[0210] Findings of the compounds of the present invention in Phase IIB clinical trials have demonstrated that the compounds of the present invention generate additional collagen production and fibrosis, filling in the lines and wrinkles at an accelerated pace.

[0211] Completed Phase IIB clinical trial (after CO2 fractional laser) The clinical trial methodology used (n=40) mimicked a standardized burn created by fractionated CO2 laser treatment over the entire chest. The double-blind / placebo-controlled clinical study was conducted by a third party.

[0212] Wrinkles Figure 18 details the percentage of patients in each of the compound of the invention and vehicle groups who achieved a 1 or more point improvement in wrinkle score between baseline and day 28 (Fitzpatrick-Goldman Classification). Chi-square test on unadjusted proportions was used to determine significance, expressed as p-value.

[0213] 85% of responders achieved a wrinkle score of 1 or greater (33% improvement) in the compound of the invention group compared to the placebo group (only 50% of responders achieved a wrinkle score of 1 or greater).

[0214] P < 0.04 for 70% variance using chi-square or Fisher exact value.

[0215] Elastosis At day 28, 75% of respondents in the active group achieved an elastosis improvement score of 3 or greater (33% improvement) compared with only 35% of respondents in the placebo group (114% variance P < 0.011).

[0216] Figure 19 details the percentage of patients in each of the compound of the invention and vehicle groups who achieved a 3 or greater improvement in elastosis score between baseline and day 28 (Fitzpatrick-Goldman Classification). Chi-square tests on unadjusted proportions were used to determine significance, expressed as p-values.

[0217] Example 17 In this example, the efficacy of the compounds of the present invention is tested against a further product of the art, published as US 9,956,245 B2, sold under the trademark Woulgan™, which the inventors consider to be the most relevant β-glucan patent publication for wound healing.

[0218] As shown in FIG. 20, Woulgan™ produced 8.9-fold greater immune response compared to the control, as measured by an assay measuring TNFα response from human harvested macrophage cells. This is a measure of immune reaction or stimulation. In contrast, TR-987 labeled with the compound of the present invention assayed a 25.4-fold greater response for the compound of the present invention versus the same vehicle control. This means that the compound of the present invention is approximately 3-fold more potent than Woulgan™ gel, and is much more effective than conventional compounds. Note that the concentration of Woulgan™ was 2% and the concentration of the compound of the present invention was (0.1%). The compound of the present invention was present at 1 / 20th the concentration of Woulgan™ gel, yet produced 3-fold greater immune response, meaning that it is a 60-fold more potent molecule in generating an immune response.

[0219] It will be understood that the embodiments of the polysaccharide compounds, their methods of use and preparation have been described by way of example only, and that modifications and additions can be made without departing from the scope of the claims herein.

Claims

A method for producing an isolated biological polysaccharide compound, wherein: The isolated biological polysaccharide compound is: A glycosyl bond containing 65-95% by weight of 1:3-linked glucopyranosyl residues and 5-25% by weight of 1:6-linked glucopyranosyl residues; A purity of 85-100% of β-D-glucan; A molecular weight of 0.5-2.2 MDa; and A TNF-α cytokine response in a human bioassay that is at least 1.5 times greater than the TNF-α cytokine response of a negative control in the human bioassay; Including, The isolated biological polysaccharide compound is essentially insoluble in an aqueous solution; The method is: Selecting yeast cells; Lysing the yeast cells and collecting cell wall fragments; Acidifying the cell wall fragments and then heating to remove mannan and additional chitin; Performing phase separation using a solvent to remove additional mannan and chitin along with proteins, glycogen, and lipids; Separating the solvent and other non-polysaccharide compounds via boiling and drying; and Performing a step of rinsing with water at least once after dissolution and before acidification Including, a method. The method according to claim 1, further comprising rinsing with water at least once after said acidification and before said phase separation. The method according to claim 1, further comprising changing the pH and washing with alcohol after acidification and before said phase separation. The method according to claim 3, wherein the pH drops to 4.0, and the alcohol is washed, then the pH rises to 9.0, and the alcohol is washed, then the pH is adjusted to 7.0, and the alcohol is washed. The method according to claim 1, further comprising further purifying through at least one additional series of solvent rinsing, alcohol rinsing, and optionally additional water rinsing before redrying the final product after separation of the solvent and other non-polysaccharide compounds via boiling and drying. The method according to claim 1, wherein the yeast cells are selected from the species Saccharomyces cerevisiae. The method according to claim 1, wherein the lysing occurs via an alkali treatment or a heat treatment or both treatments. The method according to claim 3, wherein the alcohol used is selected from one or more lower alcohols. The method according to claim 3, wherein the alcohol used is selected from methanol, ethanol, propanol, and combinations thereof. The method according to claim 1, wherein the solvent used is: an organic solvent; and a non-polar solvent; and has a specific gravity greater than 1.

0. The method according to claim 1, wherein the solvent is selected from methyl chloroform, chloroform, dichloromethane, tetrachloroethane, carbon tetrachloride, ethyl acetate, and combinations thereof. The method according to claim 1, wherein the phase separation using the solvent is completed at room temperature and neutral pH. The method according to claim 1, wherein after separation of the solvent and the other non-polysaccharide compounds via boiling and drying, the resulting dried polysaccharide is further purified through at least one additional series of solvent rinsing, alcohol rinsing, and optionally at least one additional series of water rinsing before redrying the final product. The method according to claim 13, wherein at least one additional series of said solvent rinsing and said alcohol rinsing is performed using a solvent selected from methyl chloroform, chloroform, dichloromethane, tetrachloroethane, carbon tetrachloride, ethyl acetate and combinations thereof, and an alcohol selected from methanol, ethanol, propanol and combinations thereof. The method according to claim 13, wherein at least one additional series of said water rinsing is completed using water at a temperature above 50 °C. The isolated biological polysaccharide compound produced by the method is 1:4-linked glucopyranosyl residues; 3:4-linked glucopyranosyl residues; 2:3-linked glucopyranosyl residues; 3:6-linked glucopyranosyl residues; 2:6- and 4:6-linked glucopyranosyl residues; 3:4:6-linked glucopyranosyl residues; and / or terminal-linked glucopyranosyl residues and is further characterized by a compound containing one or more additional side chains selected from one or more of the foregoing. The isolated biological polysaccharide compound produced by the method is 2-6% 1:4-linked glucopyranosyl residues; 0.01-0.5% 3:4-linked glucopyranosyl residues; 0.5-4% 2:3-linked glucopyranosyl residues; 3-10% 3:6-linked glucopyranosyl residues; 2:6 and 4:6 linked glucopyranosyl residues 0.2 - 1%; 3:4:6 linked glucopyranosyl residues 0.01 - 0.5%; and Terminal linked glucopyranosyl residues 2 - 8% The method according to claim 1, further characterized by a compound comprising an additional side chain containing the same. **Claim 18** A medicament for treating the skin of a patient in need thereof, comprising an isolated biological polysaccharide compound, A vehicle comprising a therapeutically effective amount of said isolated biological polysaccharide compound is topically applied to the wound site, Said isolated biological polysaccharide compound Contains glycosidic linkages comprising 65 - 95% by weight of 1:3 linked glucopyranosyl residues and 5 - 25% by weight of 1:6 linked glucopyranosyl residues; and A purity of 85 - 100% β-D-glucan; and A molecular weight of 0.5 - 2.2 MDa; and The TNF-α cytokine response in said human bioassay is at least 1.5 times greater than the TNF-α cytokine response of the negative control in the human bioassay Including; and Said isolated biological polysaccharide compound is essentially insoluble in aqueous solution, a medicament. **Claim 19** The medicament according to claim 18, wherein the vehicle is a gel composition. **Claim 20** The medicament according to claim 19, wherein the gel is an aqueous gel, and the isolated biological polysaccharide compound is in the form of microparticles and is suspended in the aqueous gel. **Claim 21** The medicament according to claim 19, wherein the gel composition has a viscosity exceeding 3000 cps. **Claim 22** The medicament according to claim 19, wherein the gel composition does not contain a chemical preservative. **Claim 23** The medicament according to claim 18, wherein the vehicle further comprises 0.05 - 1.5% by weight of the isolated biological polysaccharide compound. **Claim 24** The medicament according to claim 18, wherein the vehicle further comprises 0.1 - 1.0% by weight of the isolated biological polysaccharide compound. **Claim 25** The medicament according to claim 18, wherein the vehicle is applied to the wound site as a layer 1 - 5 mm thick, and over time, said layer is absorbed by the skin. **Claim 26** The medicament according to claim 18, wherein the vehicle is topically applied to chronic wounds. **Claim 27** The medicament according to claim 26, wherein the chronic wound is an ulcer. **Claim 28** The medicament according to claim 27, wherein the ulcer is a venous leg ulcer. **Claim 29** The medicament according to claim 18, which is used topically as a cosmetic treatment for the skin.

30. The pharmaceutical according to claim 29, which is used for improving healed elastosis cutis and wrinkles.

31. The pharmaceutical according to claim 18, wherein the vehicle is used for the treatment of fractionated skin and / or resurfacing of fully ablatable skin.

32. The pharmaceutical according to claim 18, wherein the vehicle is used as an auxiliary gel that is topically applied after treatment to promote benefits to the skin texture.

33. The pharmaceutical according to claim 18, wherein the molecular weight is 0.5 to 1.7 MDa.

34. A glycosyl bond comprising 65 to 95% by weight of 1:3-linked glucopyranosyl residues of an isolated biological polysaccharide compound and 5 to 25% by weight of 1:6-linked glucopyranosyl residues of the isolated biological polysaccharide compound; A purity of 85 to 100% of β-D-glucan; A molecular weight of 0.5 to 2.2 MDa; A TNF-α cytokine response in a human bioassay that is at least 1.5 times greater than the TNF-α cytokine response of a negative control in the human bioassay; and 2 to 6% of 1:4-linked glucopyranosyl residues of the isolated biological polysaccharide compound; 0.01 to 0.5% of 3:4-linked glucopyranosyl residues of the isolated biological polysaccharide compound; 0.5 to 4% of 2:3-linked glucopyranosyl residues of the isolated biological polysaccharide compound; 3 to 10% of 3:6-linked glucopyranosyl residues of the isolated biological polysaccharide compound; 0.2 to 1% of 2:6- and 4:6-linked glucopyranosyl residues of the isolated biological polysaccharide compound; 0.01 to 0.5% of 3:4:6-linked glucopyranosyl residues of the isolated biological polysaccharide compound; and 2 to 8% of terminal-linked glucopyranosyl residues of the isolated biological polysaccharide compound An additional side chain containing An isolated biological polysaccharide compound, comprising The isolated biological polysaccharide compound is insoluble in an aqueous solution. Isolated biological polysaccharide compound.

35. The isolated biological polysaccharide compound according to claim 34, wherein the isolated biological polysaccharide compound is derived from yeast cells of the Saccharomyces cerevisiae species.

36. The isolated biological polysaccharide compound according to claim 34, which is β-D-glucan with a purity of 90 - 100%.

37. The isolated biological polysaccharide compound according to claim 34, further comprising a protein content of 3% by weight or less.

38. The isolated biological polysaccharide compound according to claim 34, further comprising a protein content of 0.3% by weight or less.

39. The isolated biological polysaccharide compound according to claim 34, further comprising a lipid content of 3% by weight or less.

40. The isolated biological polysaccharide compound according to claim 34, further comprising a lipid content of 0.3% by weight or less.

41. The isolated biological polysaccharide compound according to claim 34, wherein the isolated biological polysaccharide compound is sterile and further comprises the absence of pathogens in addition to a microbial count of less than 10 cfu / g.

42. The isolated biological polysaccharide compound according to claim 34, wherein the isolated biological polysaccharide compound is in particulate form.

43. The isolated biological polysaccharide compound according to claim 42, wherein the particle size in particulate form is less than 40 μm.

44. The isolated biological polysaccharide compound according to claim 34, which is storage stable for at least one year after storage at ambient temperature.

45. The isolated biological polysaccharide compound according to claim 34, wherein the isolated biological polysaccharide compound is incorporated into a vehicle configured to be topically applied to a wound site, and the vehicle comprises a therapeutically effective amount of the isolated biological polysaccharide compound.

46. The isolated biological polysaccharide compound according to claim 45, wherein the vehicle is a gel.

47. The isolated biological polysaccharide compound according to claim 46, wherein the gel is an aqueous gel, the isolated biological polysaccharide is in particulate form, and is suspended in the gel.

48. The isolated biological polysaccharide compound according to claim 45, wherein the vehicle comprises 0.05 - 1.5% by weight of the isolated biological polysaccharide compound.

49. The glycosyl linkage is 65 - 85% by weight of 1:3-linked glucopyranosyl residues of the isolated biological polysaccharide compound and 15 - 25% by weight of 1:6-linked glucopyranosyl residues of the isolated biological polysaccharide compound, of the isolated biological polysaccharide compound according to claim 34.

50. The isolated biological polysaccharide compound according to claim 34, which is β-D-glucan with a purity of 95 - 100%.

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