Method for treating and / or preventing undesirable sequelae of turbinectomy or functional endoscopic sinus surgery

Abstract

Endoscopic surgery targeting the paranasal sinuses and turbinates helps manage the symptoms of chronic rhinosinusitis and other conditions. When modern techniques are used, postoperative bleeding, adhesion formation, and inadequate wound healing are the most common complications. This disclosure provides for the use of PuraSinus, a self-assembling peptide matrix, which significantly reduces the incidence of these postoperative complications, including the prevention of adhesion formation and the assistance of hemostasis. The present invention relates to a method for treating damaged tissue in the nasal cavity in order to promote rapid healing and inhibit the formation of intranasal adhesions (so-called "synechiae").

Description

Technical Field

[0001] (Priority) This application claims the priority of U.S. Provisional Patent Application No. 63 / 341,447, filed on May 13, 2022, the entire content of which is incorporated herein by reference.

[0002] (Sequence Listing) This application includes an XML sequence listing submitted electronically, the entire content of which is incorporated herein by reference. This sequence listing, created on April 12, 2023, is named 3DM-21-01-TRB-PCT_SL.xml and is 30,519 bytes in size.

[0003] (Field of the Invention) The present invention relates to a method for treating damaged tissue in the nasal cavity to promote rapid healing and inhibit the formation of nasal adhesions (so-called "synechiae"). More particularly, the present invention relates to a medical gel composed of self-assembling peptides that is applied during surgery to the nasal turbinates to inhibit adhesion formation, promote more rapid hemostasis, and promote more rapid and complete wound healing.

Background Art

[0004] (Background of the Invention) Endoscopic surgery targeting the paranasal sinuses and turbinates helps manage the symptoms of chronic rhinosinusitis. Turbinate surgery can also be performed in the context of cosmetic rhinoplasty. When using modern techniques, postoperative bleeding, adhesion formation, and inadequate wound healing are the most common complications. This disclosure is based, at least in part, on the discovery that the application of the self-assembling peptide RADA16 matrix dramatically reduces the incidence of these postoperative complications in a sheep model of chronic rhinosinusitis. Previous reports of using RADA16 in humans have suggested its effectiveness in nasal surgery for this purpose (M.F. Lee et al., "A novel hemostatic agent based on self-assembling peptides [RADA16] in the setting of nasal endoscopic surgery, a case series", Int. J. Surg. Case Rep. 41:461-464 (2017)). Further studies on one human patient have successfully used RADA16 applied during surgery to prevent re-adhesion of surgically separated nasopharyngeal / velar stenosis in the patient resulting from radiotherapy (E. Wong et al., Int. J. Surg. Case Rep. 70:227-229 (2020)). However, these human studies were experimental and did not definitively establish the effectiveness and mechanism of action of RADA16, particularly due to the lack of histochemical examination of postmortem tissues, which is not available in human studies. Furthermore, these studies addressed turbinate surgery, but they did not address the use of RADA16 (SEQ ID NO: 1) in other nasal surgical procedures (e.g., sinus surgery and septoplasty), nor did they address the use of other SAPs for any type of endoscopic intranasal surgery.

[0005] Over the past few decades, the innovation of endoscopic nasal surgery targeting the turbinates and paranasal sinuses has been fundamental in managing chronic rhinosinusitis that is resistant to medical treatment (R. Giger et al., Am. J. Rhinol. 17(6):327-333 (2003)).

[0006] Currently, the use of tamponade through the insertion of nasal packing is the "gold standard" in the United States and many other jurisdictions, but this sacrifices patient comfort. Hemostatic agents using gelatin matrix-thrombin tissue and chitosan gel have been verified for use in endoscopic sinus surgery (R.K. Chandra et al., Am. J. Rhinol. 17(1):51 - 5(2003); R. Valentine et al., Am. J. Rhinol. Allergy, 24(1):70 - 75(2010)). However, nasal packing and current hemostatic agents are associated with an increased rate of adhesion formation (M.S. Maccabee et al., Am. J. Rhinol. (2003) 17(4):203 - 207; R.K. Chandra, et al., Am. J. Rhinol. (2005) 19(3):240 - 3).

[0007] Therefore, there remains a need to develop new and improved methods for managing the negative sequelae of nasal surgery, particularly for reducing or preventing adhesions in turbinectomy.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0009] (Summary of the Invention) The present invention is partially based on the following research. 30 healthy sheep were surgically treated between 2018 and 2019 to create uniform nasal mucosal damage on the middle turbinate and the opposing nasal septum. Commercially available versions of self - assembling peptides (RADA16; PuraSinus; 3dmatrix.com / us / products / purasinus / ), commercially available versions of gelatin / human thrombin products (FLOSEAL), and untreated control sites were randomly assigned to alternating lesions. The primary outcome of intraoperative hemostasis was recorded, and adhesion formation and wound healing were evaluated under sedation at 2 weeks and at 6 weeks by in - situ post - mortem examination and histological examination.

[0010] The intraoperative hemostasis time was improved compared to the control wounds with RADA16 and gelatin-thrombin (FLOSEAL) (139.7 ± 56.2 seconds, 145.4 ± 58.1 seconds, and 224.0 ± 69.9 seconds, respectively; p < 0.0001 for both comparisons). The adhesion score at 2 weeks (maximum 4 points) was similar between the control (2.9 ± 1.8 points) and gelatin-thrombin (3.1 ± 1.6) wounds (p > 0.05), but was decreased by 91% compared to the control and 92% compared to the gelatin-thrombin treatment at the RADA16 site (0.3 ± 0.6 points; p < 0.0001 for both comparisons). The adhesion score at 6 weeks was similar between the control (1.1 ± 1.7) and gelatin-thrombin (1.7 ± 2.0 points) wounds (p > 0.05), but was decreased by 100% in the RADA16-treated wounds. Adhesions were less at 2 weeks at the RADA16-treated site (20%) compared to gelatin-thrombin (80%) and control (75%; p < 0.01), and were 0%, 50%, and 35% (p < 0.01), respectively, at 6 weeks. RADA16 was associated with a significantly lower 6-week histopathology score caused by a decrease in submucosal fibrosis and angiogenesis.

[0011] Regarding this sheep model of nasal surgery, the use of both PuraSinus and FLOSEAL was associated with improved hemostasis compared to the control. In the PuraSinus group alone, there was a significantly reduced adhesion formation at both 2 weeks and 6 weeks. Histopathological findings also suggest enhanced mucosal regeneration in the PuraSinus group. To further confirm these findings in humans, a prospective clinical trial is required (see, for example, M.F. Lee et al., A novel hemostatic agent based on self-assembling peptides [RADA16] in the setting of nasal endoscopic surgery, a case series, Int.J.Surg.Case Rep. 41:461~464 (2017)). The present invention is further based on the insight that this sheep study provides an experimental basis for the treatment, prevention and / or improvement of the negative sequelae of turbinectomy by using alternative self-assembling peptides having quite different chemical compositions, tensile properties, and gelling characteristics, particularly under in vivo conditions. RADA16 and gelatin-thrombin similarly promoted hemostasis in this sheep endoscopic sinus surgery model, but only RADA16 reduced postoperative adhesion formation at 2 weeks and there were no adhesions at 6 weeks. Histological examination suggested that RADA16 enhanced mucosal regeneration.

[0012] The present invention is based, at least in part, on a retrospective clinical study of 94 human adult patients who underwent various functional endoscopic sinus surgeries (FESS), including some patients who underwent turbinectomy and / or septoplasty as indicated for chronic rhinosinusitis (CRS). Intraoperative application of RADA16-I SAP (PuraSinus at 2.5% in water; (SEQ ID NO: 1)) to the surgical wound resulted in effective hemostasis in most patients in the absence of other hemostatic agents and devices. The absence of adhesions (also known as synechiae) was reported in nearly 90% of these cases at the second of two follow-up examinations for each patient. This study included human patients with a more diverse range of characteristics and sinus pathologies than other studies. This study demonstrates the utility of SAP in inducing hemostasis, promoting wound healing, preventing adhesions during longer-term recovery (the incidence of adhesions generally increases over time after surgery), and the absence of pain and patient discomfort during recovery. Also, no revision surgeries were indicated in this study.

[0013] Accordingly, the present invention provides alternative peptides (e.g., IEIK13, KLD12, etc.) for the management of the above conditions.

[0014] Briefly, in certain embodiments, the present invention includes a method of reducing negative sequelae from endoscopic turbinectomy. The method includes applying an effective amount of one or more self-assembling peptide solutions, the self-assembling peptide solution being (a) at least 65 weight / volume % purity and / or a concentration between 1.5 weight / volume % and 3.0 weight / volume % in water or physiological buffer, of SEQ ID NO: 1 and (b) at least 70 weight / volume % major peptide purity and a concentration between 1.5 weight / volume % and 3.5 weight / volume % of the total peptide concentration in water or physiological buffer, selected from SEQ ID NO: 3, the application being performed during or immediately after endoscopic turbinectomy, and the SAP solution being applied through a catheter to the surgically affected area in the turbinate, thereby contacting the wound to form a transparent absorbent adhesive gel. In some embodiments, healing over several weeks after endoscopic turbinectomy is improved in one or more of the following observable criteria: A. Reduction of epithelial erosion B. Reduction of inflammatory infiltrates C. Reduction of submucosal fibrosis D. Improved angiogenesis E. Presence or absence of seromucous glands F. Presence or absence of goblet cells and improves in one or more of the following. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] (Detailed Description of the Invention) (Characterization of Self-Assembling Peptides for Use in the Method of the Present Invention) PuraSinus (SEQ ID NO: 1) is the peptide Ac-RADARADARADARADA-CONH at approximately 2.5% in water 2It is composed of RADA16 (SEQ ID NO: 1) and potentially its truncated fragments. IEIK13 (SEQ ID NO: 2), another SAP that exhibits different gelation characteristics predicted among SAPs with different chemical structures, showed improved utility in achieving hemostasis in another type of animal model relevant to this application. As reported by S. Katsuyama et al., Minimally Invasive Therapy & Allied Technologies, 29(5):283 - 292 (2020), IEIK13 (referred to as TDM-623 in the reference) forms a harder gel (i.e., has a higher storage modulus G’) compared to RADA16 (referred to as TDM-621 in the reference) when exposed to physiological conditions, and this correlated with its product improving hemostasis when applied to punch hole injuries of the liver in pigs. Although reporting a statistically significant improvement in hemostasis compared to RADA16, these authors also reported that there was no inflammatory cell infiltration due to the presence of RADA16 gel or IEIK13 gel in the wound after application of the SAP solution to individual wounds. This is a good sign for other in vivo and clinical applications of RADA16 and IEIK13, and examples of such applications include the use of IEIK13 in preventing undesirable sequelae in turbinectomy and other types of endoscopic sinus surgeries.

[0017] The use of D - amino acid - containing SAPs (including the SAPs of RADA16 and IEIK13, KLD12 (SEQ ID NO: 2) and QLEL12 (SEQ ID NO: 4)) for intraoperative treatment of human and animal patients who have undergone turbinectomy or other types of endoscopic sinus surgeries is included in the present invention, particularly when a slower rate of in vivo degradation and / or absorption may be desirable.

[0018] In a report directly comparing RADA16 (SEQ ID NO: 1; TDM-621 in the paper) and IEIK13 (SEQ ID NO: 3; TDM-623 in the paper), although the concentration of the IEIK13 peptide solution was not shown, IEIK13 was found again to form a harder hydrogel matrix in vitro and in vivo when exposed to physiological conditions (pH of about 7.4 and at least low ion (e.g., Na + and / or K + ) concentrations (H. Masuhara et al., Anns. Thorac. Cardiovasc. Surg. 2012, 18(5):444 - 451)), and IEIK13 (SEQ ID NO: 3) showed a statistically significant improvement compared to RADA16 (SEQ ID NO: 1) in achieving hemostasis in punch - hole injuries of the liver of pigs with blood leakage. Ejected wounds were excluded from the analysis because neither gel remained at the wound (S. Katsuyama et al., "Novel, infection - free, advanced hemostatic material: physical properties and preclinical efficacy", Minimally Invasive Therapy & Allied Technologies, 29(5):283 - 292 (2019)). These researchers also stated that IEIK13 (SEQ ID NO: 3) can be stored at room temperature and is easier to handle than RADA16, and like RADA16 (SEQ ID NO: 1), IEIK13 (SEQ ID NO: 3) can be applied to the resection area through a syringe or an endoscopic catheter, which makes IEIK13 (SEQ ID NO: 3) suitable for use in surgeries such as turbinate formation and other endoscopic nasal surgeries. This latter feature is common to the SAP disclosed and claimed in the present application.

[0019] In some embodiments, the SAP is of formulas I - IV: ((Xaa neu -Xaa + )) x (Xaa neu -Xaa - ) y ) n (I) ((Xaa neu -Xaa - ) x (Xaa neu -Xaa + ) y ) n (II) ((Xaa + -Xaa neu ) x (Xaa - -Xaa neu ) y ) n (III) ((Xaa - -Xaa neu ) x (Xaa + -Xaa neu ) y ) n (IV) comprises an amino acid residue sequence according to one or more of the following, wherein Xaa neu represents an amino acid residue having a neutral charge; Xaa + represents an amino acid residue having a positive charge; Xaa - represents an amino acid residue having a negative charge; x and y are integers independently having a value of 1, 2, 3, or 4; and n is an integer having a value of 1 to 5.

[0020] In some embodiments, a particular peptide for use in the methods of the present invention can be selected from one or more of the peptides listed in Table 1 below. [Table 1]

[0021] (Peptide concentration) - According to one or more embodiments, the rheological properties of the peptide compositions described previously (U.S. Patent No. 10,654,893) can be controlled by the selection of the peptide concentration and / or through adjacency, as may be particularly preferred for a particular adaptation or use of the composition, for example, by the selection of the peptide concentration.

[0022] For a majority of SAPs, the hardness of the composition has been shown in vitro to increase substantially linearly with peptide concentration. Further, as previously described (U.S. Patent No. 10,654,893), certain peptide compositions exhibited thixotropic properties beyond a critical stress level. Considering that synthetic SAPs are commercially supplied sterilized in water within prefilled syringes, thixotropy occurs when the SAP solution is delivered from the syringe to the application site during surgery, where the SAP contacts body fluids (e.g., blood), whereby the delivered SAP self-organizes into a nanofiber matrix similar to the extracellular matrix, which is considerably harder than the gel inside the syringe in the supplied state.

[0023] In vitro, the rheological properties achieved at a specific peptide concentration vary depending on the entity of the peptide. For example, the storage modulus G’ of 1.5% KLD12 (SEQ ID NO: 2) in water was found to be approximately 350 Pa, similar to the storage modulus G’ of 2.5% RADA16 (SEQ ID NO: 1) in water under the same test conditions. The storage modulus G’ of 1% IEIK13 (SEQ ID NO: 3) in water (about 700 Pa) was similar to the storage modulus G’ of 2.5% KLD12 (SEQ ID NO: 2) in water under the same test conditions and was found to be higher than the storage modulus G’ of 2.5% RADA16 (SEQ ID NO: 1) in water (about 350 Pa) (U.S. Patent No. 10,654,893 - Tables 3 and 3A). Overall, the order of rheological strength among these compositions is IEIK13 (SEQ ID NO: 3) > KLD12 (SEQ ID NO: 2) > RADA16 (SEQ ID NO: 1), and thus, when the peptide concentration in water was the same in each case, the composition of IEIK13 (SEQ ID NO: 3) exhibited a greater rheological strength than that shown by the composition of KLD12 (SEQ ID NO: 2), and in turn, this composition of KLD12 (SEQ ID NO: 2) exhibited a greater rheological strength than that shown by the composition of RADA16 (SEQ ID NO: 1).

[0024] In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is at least 0.05%, at least 0.25%, at least 0.5%, at least 0.75%, at least 1.0% or more. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, or less. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is in the range between about 0.5% and about 3%. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is in the range between about 0.5% and about 2.5%. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is in the range between about 1% and about 3%. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is in the range between about 1% and about 2.5%. In some embodiments, the peptide concentration in the peptide composition for use in accordance with the present invention is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, or more. In some specific embodiments, when the peptide is RADA16 (SEQ ID NO: 1), the peptide concentration in the peptide composition of the present invention is in the range of about 0.05% to about 5%.

[0025] In some specific embodiments, when the peptide is KLD12 (SEQ ID NO: 2), the peptide concentration in the peptide composition of the present invention is in the range of about 0.05% to about 5%.

[0026] In certain specific embodiments, when the peptide is IEIK13 (SEQ ID NO: 3), the peptide concentration in the peptide composition of the present invention is in the range of about 0.05% to about 2.5% in water, considering that if the concentration exceeds this range in water, only this peptide may be too viscous to be useful (see U.S. Patent No. 10,654,893, Table 1 therein). However, when other components (e.g., bioactive peptides and / or pharmaceuticals) are added to the IEIK13 (SEQ ID NO: 3) solution and / or bound to the peptide, the rheological properties of the solution change such that administration of higher concentrations (e.g., 5% or more) of SAP in the solution may be useful and advantageous for various indications.

[0027] (Hydrogel formation of SAP when exposed to in vivo conditions or in vivo-like conditions) - In the present invention, the SAP solution is applied to the surgical site at the clinical site through a syringe equipped with a nozzle having a small opening and / or an endoscopic catheter. When the SAP solution is applied (rather than injected) to the target site in vivo, the SAP self-organizes at that site into nanofibers that form a hydrogel matrix, thereby causing or contributing to rapid hemostasis by occluding the blood vessels of the tissue during bleeding or leakage and remaining in contact with or integrated with the wound tissue at that site for a certain period of time. This period varies according to the type, severity, location and extent of the wound, as well as the entity of a single SAP or multiple SAPs, and their concentrations applied, the amount applied, as well as any other components constituting the SAP and the entity, form and concentration of any other components constituting the solution to which the SAP is applied. This hemostatic effect does not depend on the patient's own coagulation mechanism as shown by numerous studies. For example, in a pig model of liver punch biopsy, using a self-assembling peptide called AC5 in the paper to apply to the wound was effective for hemostasis in both heparin-treated and non-heparin-treated animals (D. Csukas et al., Nanomedicine: Nanotechnology, Biology and Medicine, 2015, 11(8): 2025~2031).

[0028] In water, 1% each of RADA16 (SEQ ID NO: 1), KLD12 (SEQ ID NO: 2), and IEIK13 (SEQ ID NO: 3) SAPs showed a multiple increase in storage modulus G’ after treatment in DMEM (Dulbecco's Modified Eagle Medium) to mimic in vivo conditions. The increase in G’ under the same conditions was 43.5-fold, 23.9-fold, and 7.4-fold, respectively, after DMEM treatment compared to before treatment for RADA16 (SEQ ID NO: 1), KLD12 (SEQ ID NO: 2), and IEIK13 (SEQ ID NO: 3). Among the samples treated with DMEM, IEIK13 (SEQ ID NO: 3) was shown to have the highest storage modulus of 5314 Pa (U.S. Patent No. 10,654,893; Figures 5A and 5B). This increased hardness of IEIK13 (SEQ ID NO: 3) compared to RADA16 (SEQ ID NO: 1) has been shown in situ (Caravasili et al., ACS Biomater. Sci. Eng. 3(12):3386 - 3394 (2017)) and in vivo (S. Katsuyama et al., Minimally Invasive Therapy & Allied Technologies, 29(5):283 - 292 (2020)).

[0029] PuraSinus (SEQ ID NO: 1) of the medical device applied to the surgical site in the claimed method is an aqueous solution of a self - organizing absorbable peptide currently used in bleeding after endoscopic resection, which has been found to provide effective hemostasis and is easy and safe to use. As a soluble and transparent gel that can be applied through a nozzle or an endoscopic catheter, the ease of application of this gel is suitable for use deep in the nasal cavity. As shown by the studies described herein, the application of PuraSinus ((SEQ ID NO: 1) (3 - D Matrix, Ltd. or 3 - Matrix, Inc.)) at the surgical site after surgery shows improved healing compared to a control group not treated with a healing agent after turbinectomy and compared to subjects treated with FLOSEAL (Baxter Healthcare).

[0030] Currently, in Australia, PuraStat (PuraSinus or PuraGel; RADA16-I (SEQ ID NO: 1)) is approved for use in exudative bleeding from the blood vessels and parenchyma of solid organs, vascular anastomoses, and the small blood vessels and capillaries of the gastrointestinal tract (ARTG registration 267486). The self-assembling peptides (SAPs) disclosed herein have shown improved outcomes after this type of surgery in studies developed in accordance with the international standard ISO 10993-1, "Biological evaluation of medical devices" (2016). All staff and facilities conducted this study in accordance with WHO Good Laboratory Practice.

[0031] In another embodiment of the invention, Ac-IEIKIEIKIEIKI-NH, which is an SAP 2(IEIK13 (Accession No. 3; also known as TDM-623)) exhibits different gelation properties in vitro compared to RADA16 (TDM-621 in this study; (Accession No. 1)) and achieved improved hemostasis in vivo in a porcine model of punch hole injury to the liver (Katsuyama S. et al., "Novel, infection-free, advanced hemostatic material: physical properties and preclinical efficacy", Minimally Invasive Therapy & Allied Technologies, 29:5, 283-292 (2020)). For example, in a solution (assumed to be 2.5 wt / vol% SAP), the reported median storage modulus G' of RADA16 was 384 Pa and the reported median loss modulus G'' was 24 Pa, while in those studies, the storage modulus of IEIK13 (concentration unknown) was 1145 Pa and the loss modulus was 116 Pa. These differences were statistically significant. After hydrogel formation in DMEM in vitro, the storage modulus and loss modulus of RADA16-I (Accession No. 1) and IEIK13 showed substantial increases. This observed increase is consistent with many other studies. Furthermore, these values were significantly higher after DMEM treatment than before (p <.0001). The viscosity of IEIK13 was higher compared to RADA16 ((Accession No. 1); both before and after gelation) and appeared to correlate with improved short-term hemostasis, which was likely due in part to better retention of the gel in the wound in in vivo studies. This animal study (S. Katsuyama et al.) appeared to last only 5 minutes. This study was not intended to investigate healing or longer-term hemostasis at the wound site, nor to suggest the advantages of IEIK13 SAP compared to RADA16-I (Accession No. 1) SAP in wound treatment.

[0032] In US Patent Application Publication No. 2016 / 0317607, further findings of the properties of PuraSinus (referred to as PuraStat (SEQ ID NO: 1) in this application) having a major peptide content exceeding 65% to 70% reveal several advantages of these peptides. Tested solutions containing less than 65% to less than 70% pure peptides were not observed to self-organize into a gel when exposed to a near-neutral pH. Thus, in a preferred embodiment of the present invention, at least 65%, 70%, 75%, 80%, 85% or more of the SAP found and used in the administered solution is the full-length peptide, and the remainder is less than full-length versions due to synthetic impurities and / or degradation of the full-length peptide.

[0033] The transparency of the above SAP hydrogel is advantageous for visualization of the tissue covered by the gel in vivo and for observation of wound healing while the nanofiber matrix is still present. This advantage of transparency is described in European Patent Application Publication No. 2581097, but the wound dressing disclosed in this application is not suitable for endoscopic administration.

[0034] In some embodiments, the amino acid residues in the SAP may be naturally occurring amino acid residues or non-naturally occurring amino acid residues. Naturally occurring amino acids include amino acid residues encoded by the standard genetic code, as well as non-standard amino acids (e.g., amino acids having a D-configuration instead of an L-configuration, or combinations of D-amino acids and L-amino acids), and amino acids that can be formed by modification of standard amino acids (e.g., pyrrolidine or selenocysteine). Suitable non-naturally occurring amino acids include, but are not limited to, D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentylglycine (S)-2-amino-2-cyclopentylacetic acid. In other embodiments, another type of material that can self-organize is a peptidomimetic. As used herein, a peptidomimetic refers to a molecule that mimics a peptide structure. Peptidomimetics have general characteristics (e.g., amphiphilicity) similar to those of their parent polypeptide structure. Examples of such peptidomimetic substances are described in Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). Peptidomimetic substances can be classified into the following four categories: α-peptides, β-peptides, γ-peptides, and δ-peptides. Copolymers of these peptides can also be used. Examples of α-peptide peptidomimetics include, but are not limited to, N,N'-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides, and azapeptides. Examples of β-peptides include, but are not limited to, β-peptide foldamers, α-aminooxy acids, sulfur-containing β-peptide analogs, and hydrazinopep tides. Examples of γ-peptides include, but are not limited to, γ-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters. Examples of δ-peptides include, but are not limited to, alkene-based δ-amino acids and carbo peptoids (e.g., pyranose-based carbo peptoids and furanose-based carbo peptoids).

[0035] In certain embodiments, the SAP is AC5, AC5-V, AC5-G, or TK45 (also known as AC1) (see Arch Therapeutics, Inc. at www.archtherapeutics.com).

[0036] Published U.S. Patent Application No. 2021 / 0299326, which is also related to this application, observed useful effects resulting from the sterilization of SAP in water using radiation. Samples contained 2.5% PuraSinus (Ac-RADARADARADARADA-NH 2 , RADA16 (SEQ ID NO: 1)) in water, 1.3% IEIK13 (Ac-IEIKIEIKIEIKI-NH 2( SEQ ID NO: 3)) in water, and 0.15% QLEL12 (Ac-QLELQLELQLEL-NH 2( SEQ ID NO: 4)) in water. Each of these self-assembling peptides is capable of forming a hydrogel when applied to living tissue (e.g., in situ) at approximately neutral pH. Generally, the SAP concentration in water ranges from about 1 wt / vol% to about 5 wt / vol%, although this range is not exclusive. As disclosed in U.S. Patent Application Publication No. 2021 / 0299326, gamma irradiation sterilization enhanced the rheological properties of certain self-assembling peptide solutions and hydrogels (i.e., RADA16 and IEIK13) without significant degradation as expected, while other peptides (i.e., QLEL12) showed significant degradation and viscosity reduction after gamma irradiation as predicted. As is known in the art, for relatively pure SAP (where "relatively pure" means that 70% or more of the specific peptide of a given exact sequence in the preparation is full length), synthesizing and purifying substantial amounts using conventional filter sterilization is not commercially feasible because a large amount of the peptide is lost in the process.

[0037] Other observed useful effects of the sterilization of selected SAPs using radiation are of further value in the present invention. In preferred embodiments, the compositions and methods of the present invention maintain or improve desirable biological properties (e.g., hemostasis, anti-adhesion, prevention of rebleeding, anti-stenosis, tissue occlusion, storage modulus, viscosity, and tissue void filling).

[0038] In US Patent Application Publication No. 2016 / 0317607 (entitled "Purified Amphiphilic Peptide Composition and Uses Thereof"), further findings of the properties of PuraSinus (RADA16 - called PuraStat in this application) with a major peptide content exceeding 65% - 70% reveal advantages over the prior art. Regarding the above application, the affidavit of Dr. Eun Seok Gil dated April 24, 2021, reiterates the advantageous properties of a purified SAP of exactly the same sequence in which at least 75% of that peptide is full-length. Tested solutions containing less than 65% - less than 70% pure peptide were not observed to self-organize into a gel when exposed to a nearly neutral pH. Thus, it would not be useful here.

[0039] The transparency of the above-described SAP gel described in the above application is advantageous for visualization of the tissue covered by the gel in vivo and for observation of wound healing before the predicted complete absorption of the matrix in about 30 days. This advantage of transparency is described in European Patent Application Publication No. 2581097 assigned to Arch Therapeutics, Inc., but the wound dressing disclosed in this application is not suitable for endoscopic administration.

[0040] (Method of the Invention) The PuraSinus used in this study is the peptide Ac-RADARADARADARADA-NH in about 2.5% in water 2(RADA16-I (SEQ ID NO: 1)) and potentially fragments derived therefrom. As a soluble and transparent gel that can be applied through an endoscopic catheter, the ease of application of this gel is suitable for its use deep in the nasal cavity and shows improved healing at the surgical site after surgery compared to a control group not treated with a healing agent after surgery and compared to subjects treated with FLOSEAL (Baxter Healthcare).

[0041] Currently in Australia, under the Australia Register of Therapeutic Goods, PuraStat is approved for use in the vascular and parenchymal organs, vascular anastomosis, and in the treatment of exudative bleeding from small blood vessels and capillaries in the gastrointestinal tract (ARTG registration 267486). The self-assembling peptides (SAPs) disclosed herein show improved results after this type of surgery in studies developed in accordance with the international standard ISO 10993-1, "Biological evaluation of medical devices" (2016). All staff and facilities conducted this study in accordance with WHO Good Laboratory Practice.

[0042] The present invention provides a method for reducing negative sequelae after endoscopic nasal surgery in a mammal, the method comprising applying an effective amount of a self-assembling peptide (SAP) solution, the application being performed at a site of tissue damaged as a result of endoscopic nasal surgery, trauma and / or pathology in the paranasal sinuses and / or turbinates, said SAP being applied to the site through a syringe equipped with a (compatible) nozzle or through an endoscopic catheter, whereby the SAP solution forms a gel when in contact with blood or other body fluids, induces rapid hemostasis and / or reduces adhesion formation, provided that RADA16 (SEQ ID NO: 1) consisting of L-amino acids is excluded from the SAPs used in endoscopic turbinectomy. In some embodiments, the SAP is selected from the group consisting of (a) RADA16 (SEQ ID NO: 1), (b) KLD12 (SEQ ID NO: 2), (c) IEIK13 (SEQ ID NO: 3), and (d) KLDL12 (SEQ ID NO: 5). In some embodiments, the SAP solution comprises SAP that is at least 65%; 70%; 75%; 80%; 85% pure with respect to the full-length peptide compared to its truncated form. In some embodiments, the SAP comprises IEIK13 (SEQ ID NO: 3) at a concentration of 0.5% and 5%, preferably 0.5% to 3.0%. In some embodiments, the endoscopic surgery is turbinectomy, septoplasty, and / or paranasal sinus surgery. In some embodiments, the site of SAP solution application includes one or more paranasal sinus ostia, thereby preventing the formation of "blockages" or blocking synechiae that can close off the air circulation in the paranasal sinuses and create an environment for chronic paranasal sinus infections. In some embodiments, the site of SAP solution application is located in the lower part of the turbinate / septum and / or the middle part of the turbinate / septum.

[0043] The outcome of the method can be evaluated by several means, for example, by a reduction of at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of adhesion formation. In some embodiments, the healing over several weeks after endoscopic surgery is based on the following observable evaluation criteria: A. Reduction of epithelial erosion; B. Reduction of inflammatory infiltrate; C. Reduction of submucosal fibrosis; D. Improved angiogenesis; E. Presence or absence of seromucous glands; and F. Presence or absence of goblet cells is improved in one or more of.

[0044] Other aspects of the invention will be apparent to those skilled in the art based on this specification, including the examples and the appended claims.

Examples

[0045] (Example 1: SAP Hydrogel for Hemostasis and Prevention of Adhesion Formation after Turbinate Surgery in a Sheep Model of Chronic Rhinosinusitis) (Abstract) - Endoscopic surgery was performed on 30 healthy sheep to create a model for chronic rhinosinusitis (CRS) by making uniform mucosal injuries to the middle turbinate and the opposing nasal septum. The effect of endoscopic treatment of the surgical wounds with a commercially available version of self-assembling peptide (2.5% RADA16-I in water; PuraSinus; 3dmatrix.com / us / products / PuraSinus / ) was compared with the results of treatment with a gelatin / human thrombin product (FLOSEAL) and also with untreated controls. The treatment method for the surgical sites (two sites / side per sheep) was randomly selected by computer. As presented in Appendix 1, the primary outcome of intraoperative hemostasis was recorded, adhesions formation and wound healing were evaluated endoscopically under sedation at 2 weeks, and at 6 weeks by postmortem examination and histological examination in situ. The intraoperative hemostasis time was improved compared to the control wounds with RADA16 and gelatin-thrombin (139.7 ± 56.2 seconds, 145.4 ± 58.1 seconds, and 224.0 ± 69.9 seconds, respectively; p < 0.0001 for both comparisons). The adhesion score at 2 weeks (maximum 4 points) was similar between the control (2.9 ± 1.8 points) and gelatin-thrombin (3.1 ± 1.6) wounds (p > 0.05), but was decreased by 91% compared to the control and 92% compared to the gelatin-thrombin treatment at the RADA16 sites (0.3 ± 0.6 points; p < 0.0001 for both comparisons). The adhesion score at 6 weeks was similar between the control (1.1 ± 1.7) and gelatin-thrombin (1.7 ± 2.0 points) wounds (p > 0.05), but was decreased by 100% in the RADA16-treated wounds. Adhesions were less at 2 weeks in the RADA16-treated sites (20%) compared to gelatin-thrombin (80%) and control (75%; p < 0.01), and at 6 weeks were 0%, 50% and 35% respectively (p < 0.01). RADA16 was associated with a significantly lower 6-week histopathological score caused by a decrease in submucosal fibrosis and angiogenesis. Furthermore, PuraSinus was generally associated with enhanced mucosal regeneration as measured by epithelial erosion / epithelial regeneration, inflammatory infiltrates, submucosal fibrosis, angiogenesis, and regeneration of seromucous glands / goblet cells based on the histopathological score.

[0046] (Details of Sheep Research) - Endoscopic nasal surgery targeting the turbinates and paranasal sinuses is fundamental in managing the symptoms of chronic rhinosinusitis. A sheep model was selected to test the treatment of endoscopic surgical wounds in the deep paranasal sinuses using PuraSinus (2.5% RADA16-I in water; (SEQ ID NO: 1)) in comparison to a control as a model for rhinoplasty in humans, which was due to both the size of the animal and the suitability of the sheep model for comparison with this type of surgery in humans as follows. PuraSinus (ARTG: 267486; (SEQ ID NO: 1)) was supplied by 3-D Matrix Europe SAS as a synthetic SAP solution of 2.5 wt / vol% in water having a pH of approximately 2.3. When in contact with physiological tissues and / or liquids (such as blood), the pH and / or ionic strength of PuraSinus (SEQ ID NO: 1) increases, inducing self-organization of the peptide into nanofibers and forming a β-sheet network similar to the extracellular matrix. At the microscopic level, the viscous solution forms a hydrogel that seals open blood vessels, resulting in hemostasis. PuraSinus (SEQ ID NO: 1) is biocompatible, does not contain infectious agents, and is absorbed over time, although some residue may remain for more than 30 days. It is supplied sterile in a syringe and delivered through a nozzle supplied by the manufacturer. It is stored at 2°C to 8°C. In this example, FLOSEAL (ARTG: 192294) was obtained from Baxter Healthcare Pty Ltd. The FLOSEAL kit consists of a bovine-derived gelatin matrix, a human-derived thrombin component, an applicator tip, and several mixing accessories. The mixing accessories include a syringe with an integrated female luer connector, a small bowl, and a 5 mL syringe with a needle attached. The mixing accessories are included to facilitate reconstitution and mixing of the thrombin into the gelatin matrix. The applicator tip is included to facilitate delivery of FLOSEAL to the site to be treated. The gelatin matrix consists of cross-linked gelatin granules and is provided sterile and pyrogen-free in a standard disposable syringe.

[0047] The thrombin (human) component in FLOSEAL is a lyophilized, vapor-heated, solvent and surfactant-treated powder preparation that is generated from pooled human plasma, sterilized, and free of pyrogens. The sodium chloride solution is a sterilized solution free of pyrogens. After constitution of the lyophilized thrombin in the sodium chloride solution, the resulting thrombin solution contains 500 IU / mL of thrombin (human). FLOSEAL is a combination of a gelatin matrix and a thrombin component.

[0048] Thrombin must be added to the gelatin matrix prior to use. FLOSEAL is biocompatible but not necessarily free of infectious agents or allergens, and is absorbed within 6 to 8 weeks, which is consistent with normal wound healing (see FLOSEAL IFU). FLOSEAL is used as a hemostatic agent for surgical procedures that are refractory to ligation or conventional techniques. It is stored at room temperature between 2°C and 25°C.

[0049] (Animal Subjects) - Thirty previously unused healthy castrated male Merino sheep (Ovis aries) of a specific age and weight were selected for these studies. The use of the sheep model has been validated for studying the outcomes of endoscopic nasal surgery (C.L. Shaw et al., Aust. J. Otolaryngol. 4(1):23 - 26 (2001)), and extensive use in numerous references has evaluated the effectiveness of similar hemostatic aids (e.g., Valentine R. et al., Otolaryngol. Clin. North A. 42(5):813 - 28 (2009)). Alternative models (e.g., New Zealand White rabbits) have been explored in the past, but the difficulty in obtaining rabbits free of Pasturella has a risk of confounding results. This sheep is an ideal model because its internal anatomical structures (paranasal sinuses, inferior and middle turbinates) are similar to those of humans, and the size of the nasal cavity allows easy access for surgical instruments and endoscopes for postoperative monitoring. General anesthesia was performed for the entire surgical procedure in each sheep to prevent any intraoperative discomfort and / or pain that could occur. Additionally, preoperative and postoperative analgesics were used, and the sheep were monitored for pain and distress.

[0050] There are no validated available in vitro assays or computer simulation models that can mimic the complexity of postoperative adhesion formation. These studies were conducted at the USYD University Veterinary Centre Camden (Australia) and complied with all the criteria applied for the humane handling and management of animals.

[0051] (Animal Management) - The animals were housed at the USYD University Veterinary Centre Camden in accordance with their established management criteria. The animals were housed in an outdoor pasture and monitored daily. Ear tags were used to identify the animal number, test code, and surgery date.

[0052] Sheep were allocated to areas of the UVCC pasture specialized for daily feeding. Drinking water was supplied through water containers appropriate for the species or delivered through an automatic watering system. No contaminants that were predicted to affect the results of this study were present in their feed or water. The UVCC is an accredited veterinary teaching hospital, clinic, and research center. All research conducted on the premises is subject to ethical approval from the University of Sydney’s Animal Ethics Committee as required by the NSW Animal Research Act (1985).

[0053] Colleagues involved in this study were appropriately qualified and trained and had experience in large animal surgery and endoscopic nasal surgery. It was determined that the use of sedation, analgesia, or anesthesia was necessary during the normal course of this procedure. All anesthetic, analgesic, and other drugs were administered or altered at the discretion of a veterinarian or anesthetist according to standard veterinary practice and the objectives of this study. This applies to specific drugs, dosages, and dosing intervals. None of the animals were injured, became ill, or were near death and did not require care (e.g., euthanasia) prior to the end of this study.

[0054] (Surgical method) - On the day before surgery, the animals were weighed, and after surgical turbinate formation, PuraSinus was given to each nostril, FLOSEAL was given, or no treatment was given, and they were randomly assigned to each nostril by computer randomization. According to standard anesthesia procedures, each animal was not given food for 24 hours before surgery and was not given water for 8 to 12 hours before surgery. The animals were sedated with a weight-adjusted dose of intravenous diazepam. After sedation, a percutaneous jugular catheter was inserted and fixed, and induction was achieved with a weight-adjusted dose of additional intravenous diazepam and ketamine. The animals were placed in the sternal recumbent position for insertion of an appropriately sized endotracheal tube. Anesthesia was maintained by administering propofol and ketamine intravenously if necessary through inhalation of 1% to 2% isofluorane. Thereafter, each animal was administered a weight-adjusted dose of oxytetracycline, a prophylactic dose of a long-acting antibiotic, to protect against postoperative infections. The nasal cavity was prepared by packing it with surgical pledgets soaked in a 1% lignocaine-phenylephrine mixture. The middle turbinate was infiltrated with the same lignocaine / phenylephrine mixture by small-gauge needles at multiple sites throughout the entire middle turbinate. The use of vasoconstrictors and decongestants (e.g., lignocaine and phenylephrine) in both topical use and local injection is standard preoperatively in patients undergoing turbinate formation (Ahmed S. et al., J. Coll. Physicians Surg. Pak. 26(6):531-2 (2016)). The vital signs of the animals were monitored throughout the surgery.

[0055] Before making the incision, all sheep were first examined endoscopically to screen for any nasal septum deviation. Only animals with a straight nasal septum and a uniform space between the nasal septum and the middle turbinate were used.

[0056] (Surgical procedure) - A uniform-sized, 2 cm x 3 cm full-thickness mucosal lesion with the periosteum removed was created on the lower edge of the medial surface of the middle turbinate using a Medline Microdebrider. The large size of the vomer bone in the sheep serves as an anatomical landmark to standardize the location of the lesion.

[0057] Furthermore, a 2 cm x 3 cm mucosal injury of uniform size was created on the opposite side of the nasal septum. Due to the volume size of the middle turbinate in the sheep, creating a wound adjacent to the contralateral nasal septal wound enabled the inventors to create an environment prone to adhesion so that the inventors could best study the anti-adhesion properties of various products. The surgeon was not informed of the intended product for the wound before the wound was created.

[0058] Depending on the results of randomization, either 2.5% w / v PuraSinus (SEQ ID NO: 1) in water, FLOSEAL, or nothing was applied to the lesion immediately after its creation. FLOSEAL was prepared during the surgery by mixing thrombin solution and microbeads according to the manufacturer's instructions. PuraSinus (SEQ ID NO: 1) was applied in the form supplied by the manufacturer without any preparation. Both products were applied with a 20-cm applicator in a posterior-to-inferior fashion along the length of both injuries in the nasal cavity. The surgeon was not informed which product was to be applied to which wound until the surgeon handled the assigned product. Further blinding regarding the specific product to be used was not possible because the physical appearance of PuraSinus (SEQ ID NO: 1) and FLOSEAL was quite different with different application methods. 5 milliliters (ml) of either product was uniformly applied to the wound according to the intended application method of that product. In the case of PuraSinus (SEQ ID NO: 1), this was achieved through uniform application directly envisioning the state-of-the-art technology. No other hemostatic aids (e.g., electrocautery or nasal packing) were used to best evaluate the performance of the product alone.

[0059] (Intraoperative Observation) - The degree of bleeding / hemostasis was evaluated by independent blinded observers with respect to the appearance of FLOSEAL and PuraSinus (SEQ ID NO: 1), as well as the background of these two hemostatic agents. The surgical field was graded by the number of seconds it took to achieve complete hemostasis with no further blood observed draining from the nose. The time to hemostasis observed for each site is reported in Appendix 1. All suctioning was performed by carefully introducing a thin suction catheter tip in a manner that did not agitate the product within the empty space between the nasal septum and the application area.

[0060] (Recovery from Anesthesia) - After surgery, the animals were moved to the recovery area and vital signs were continuously monitored. The animals were maintained in the sternal recumbent position and the first stomach gas was allowed to escape. Once the animals were able to swallow and chew spontaneously, extubation was attempted. Once recovered, they were administered standard postoperative analgesia through a combination of opioid and NSAID, and standard postoperative antibiotics. Locally acting analgesia was avoided so as to minimally interfere with the surgical site.

[0061] (Postoperative Findings) - After recovery from anesthesia, the animals were returned to their enclosures and daily monitoring (including bloody nasal discharge, body temperature, and food and water intake) was continued for 14 days. Daily postoperatively, the animals were externally examined for the presence of bloody nasal discharge by independent observers who had no involvement in the surgery. After each examination, the external blood was cleaned and then each nostril was irrigated with 20 ml of saline. Regular irrigation of the nose and paranasal sinuses is standard postoperative care after nasal surgery.

[0062] The sheep were sedated on day 14 and re-examined endoscopically by independent observers for the presence of adhesions and graded using the following grading scheme for sheep nasal adhesions. Representative endoscopic images of the sites treated with PuraSinus (SEQ ID NO: 1) (two sites / side per sheep) are shown in Figure 1, and untreated control sites are shown in Figure 2.

[0063] The sheep was euthanized on day 42, and necropsy was performed by the same independent observer to grade the formation and extension of adhesions in the nasal cavity. Representative in situ endoscopic images of the sites treated with PuraSinus (SEQ ID NO: 1) are shown in Figure 3A. Those findings were classified according to the scheme presented in Table 2 below with respect to the degree of adhesion formation observed by endoscopic imaging at both day 14 and day 42. Exemplary endoscopic images corresponding to each grade are shown in Figure 3B. [Table 2]

[0064] (Slaughter) - On day 42, the animals were euthanized by a standard terminal procedure through intravenous injection of sodium pentobarbital. After slaughter, the mucosa of the surgically treated middle turbinate site was excised and prepared by hematoxylin and eosin staining by a veterinary histopathologist using six different wound healing markers shown in Table 3. [Table 3]

[0065] Representative tissue sample images taken from the surgical wound site 6 weeks after surgery are shown in Figures 4A and 4B for the sites treated with PuraSinus. Images of the tissue taken from the tissue treated with FLOSEAL are shown in Figures 5A and 5B. The findings by a blinded observer for the specimens derived from each of the sheep graded according to the factors listed in Table 2 are presented in Appendix 3 and summarized below. Those results suggest an improved outcome for treatment with PuraSinus compared to treatment with FLOSEAL.

[0066] (Statistical analysis) - Data are presented as mean ± standard deviation (SD), mean ± 95% confidence interval (CI), or percentage, as indicated. Continuous variables were compared among the three groups using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to adjust for multiple corrections. Categorical data were evaluated using Fisher's exact test. A two-sided p-value < 0.05 was considered statistically significant. Prism v.5.03 statistical and graphing software (GraphPad Software Inc., San Diego, CA) was used. The individual results observed are presented in Appendix 3.

[0067] (Sample size calculation) - In a previous study (J.G. Medina and S. Das, Laryngoscope, 123(1):42 - 7 (2013)), it was found that the control group (14 sites) had an adhesion rate of 86% with a 95% CI (confidence interval) of ±17.5, and the treatment group (10 sites but using chitosan hemostatic agent) had an adhesion rate of 10% with a 95% CI of ±16.5. The mean difference between those groups was 76% with a 95% CI of ±34. The mean difference was robust because all values of the 95% CI were positive. This meant that the probability of the treatment group having a lower adhesion rate than the control group was very high. However, the margin of error was very large (±34), and thus, it was difficult to determine clinical significance. The inventors calculated that 20 surgical sites (10 animals) in each group would provide a confidence interval of ±20%.

[0068] (Detailed sheep model findings) - Uniform middle turbinate injuries were successfully created in all 30 sheep between 2018 and 2019 through four groups consisting of 4, 9, 9, and 8 sheep, respectively. The large size of the nasal cavity and nasal vomer allowed the inventors to easily reproduce the standardized injury in all sheep with respect to both size and location. A total of 4 ml of the product was applied to two sites to allow sufficient coverage of the injury.

[0069] After the first surgical procedure in the pilot group, intraoperative sedation rather than general anesthesia was used for the 14-day follow-up. This option was proven to be safer for the animals and more cost-effective. All sheep survived until the 42-day target and were euthanized, followed by necropsy and histopathological examination.

[0070] (Clinical findings) - Effective compliance with routine nasal irrigation in the postoperative setting was implemented through daily irrigation with 20 ml of saline for the first 14 days. Daily observation of the sheep reported no excessive external bleeding.

[0071] The results presented in Appendix 2 regarding the observed adhesion formation can be summarized as follows:

[0072] Two weeks after surgery, the number of sites observed for each grade defined in Table 2 in each group was as follows: FLOSEAL PuraSinus Control Grade 0 4 16 5 Grade 1 0 3 1 Grade 2 1 1 0 Grade 3 1 0 0 Grade 4 14 0 14

[0073] The best results, meaning the lowest number of observed adhesion formations, were for the surgical sites treated with PuraSinus (SEQ ID NO: 1). Six weeks after surgery, the results observed regarding the degree of adhesion formation also showed a statistically significant improvement over both the untreated control and the wounds treated with FLOSEAL as follows: FLOSEAL PuraSinus Control Grade 0 10 20 13 Grade 1 2 0 2 Grade 2 0 0 0 Grade 3 0 0 0 Grade 4805

[0074] These results indicate that PuraSinus (SEQ ID NO: 1) was effective in inhibiting the formation of adhesions between the surgically traumatized tissue and the adjacent posterior nasal tissue, as compared to both FLOSEAL and the untreated control.

[0075] (Histopathological findings) - A vertical incision through the nasal dorsum and floor was performed during necropsy after endoscopic evaluation. This allowed both nasal sidewalls to be opened laterally from the nasal septum, thereby enabling easy identification and atraumatic excision of the surgically treated mucosa near the vomer bone.

[0076] Specimens were stored in 10% neutral buffered formalin and transported for hematoxylin and eosin staining and slide preparation. Exemplary specimens are shown in FIGS. 4A and 4B for PuraSinus-treated tissue and in FIGS. 5A and 5B for FLOSEAL-treated tissue. The findings for individual samples are presented in Appendix 3. The results can be summarized as follows: Epithelial erosion (EE): Number of sites per grade FLOSEAL PuraSinus Control Grade 0 457 Grade 1 483 Grade 2 665 Grade 3 615 Mean score 1.75 1.15 1.35 Inflammatory infiltrate (II): FLOSEAL PuraSinus Control Grade 0 130 Grade 1 121313 Grade 2 746 Grade 3 001 Mean score 1.30 1.05 1.35 Submucosal fibrosis (SF): FLOSEAL PuraSinus Control Grade 0 0 4 0 Grade 1 3 12 4 Grade 2 3 3 6 Grade 3 14 1 10 Average Score 2.55 1.05 2.3 Angiogenesis (A): FLOSEAL PuraSinus Control Grade 0 3 9 3 Grade 1 4 6 6 Grade 2 6 4 7 Grade 3 7 1 4 Average Score 1.85 0.85 1.60 Seromucinous Gland (SMC): FLOSEAL PuraSinus Control Grade -1 13 6 12 Grade 0 6 14 8 Grade +1 1 0 0 Average Score -0.6 -0.3 -0.6 Goblet Cell (GC): FLOSEAL PuraSinus Control Grade -1 7 8 6 Grade 0 12 6 1 Grade +1 1 6 13 Average Score -0.3 -0.1 -0.25

[0077] (Summary of Results) - The article PuraSinus and the product FLOSEAL show a statistically significant decrease in the time to hemostasis compared to the untreated control group. PuraSinus is associated with a statistically significantly lower adhesion incidence when compared to both the FLOSEAL group and the untreated control group at both 2 weeks and 6 weeks - see Figure 7.

[0078] The histopathological examination results suggest favorable outcomes for enhanced wound healing compared to the FLOSEAL group across all six evaluation criteria (epithelial erosion, inflammatory infiltrate, submucosal fibrosis, angiogenesis, seromucous glands, and goblet cells) in the PuraSinus group; see also FIGS. 4A, 4B, 5A, and 5B.

[0079] (Record) - All raw data, photographs on digital media, wet tissues, blocks, and tissue slides related to this study, as well as copies of the final reports, are kept in the coordinator's archive in accordance with WHO Good Laboratory Practice and USYD Ethics.

[0080] (Example 2: Use of SAP in functional endoscopic sinus surgery for hemostasis, wound healing, and reduction of adhesion formation) (Summary) - In a retrospective clinical trial of 94 human adult patients who underwent various functional endoscopic sinus surgeries (FESS) (including some patients who underwent turbinectomy and / or septoplasty required for chronic rhinosinusitis (CRS)), intraoperative application of RADA16-I SAP (PuraSinus at 2.5% in water; (SEQ ID NO: 1)) to the surgical wound resulted in effective hemostasis in most patients in the absence of other hemostatic agents and devices. The absence of adhesions (also known as synechiae) was reported in nearly 90% of these cases at the second of two follow-up examinations for each patient. This study included human patients with a more diverse range of characteristics and sinus pathologies than other studies. This study demonstrates the usefulness of SAP in inducing hemostasis, promoting wound healing, preventing adhesions during a longer period of recovery (the incidence of adhesions generally increases over time after surgery), and the absence of pain and patient discomfort during recovery. Also, no revision surgeries were required in this study.

[0081] (Introduction) - Functional endoscopic sinus surgery (FESS), including turbinectomy, is one of the most common surgeries performed in otolaryngology for patients with chronic rhinosinusitis (CRS) who have failed maximal medical therapy (J. Selvarajah et al., Int. J. Mol. Sci. 21(2):480(2020)). CRS affects millions of individuals and has a significant impact on quality of life (M.S. Benninger et al., Otolaryngology-Head and Neck Surgery. 129(Suppl 3):S1~32(2003)). Surgical intervention is appropriate in patients who have failed maximal medical therapy and in whom anatomical abnormalities or difficult CRS that cannot be managed conservatively are suspected (K.C. Welch and J.A. Stankiewicz, The Laryngoscope, 119(11):2258~68(2009)). This surgical approach aims to restore drainage and airflow across the affected sinuses (R.K. Weber and W. Hosemann, GMS current topics in otorhinolaryngology, head and neck surgery;14(2015)).

[0082] Considering the vascular distribution of the nasal mucosa, sinus surgery is associated with a moderate risk of bleeding, especially in inflammatory conditions (e.g., CRS) (Pant H. Otolaryngologic Clinics of North America, 49(3):655~76(2016)). As previously explained, hemostasis is necessary to prevent postoperative complications (e.g., delayed bleeding, inadequate wound healing, and adhesion formation). A series of human consecutive patients who underwent FESS were treated using these SAPs in aqueous solution. The aim of this study was to evaluate the effectiveness of PuraSinus in achieving rapid intraoperative and postoperative hemostasis and reducing adhesions after FESS.

[0083] (Study Design) - This study was a retrospective chart review of 94 human consecutive patients who underwent FESS and were surgically treated by a single surgeon at a single facility in Perth, Western Australia. Figures 6A and 6B show patient flowcharts based on the extent of surgery and outcomes. Only patients treated with PuraSinus alone (without using any other hemostatic agents) were included in this analysis. Ethical approval was obtained from the St John of Good Health Care Human Research Ethics Committee.

[0084] (Patient Population) - This study included adult human patients who underwent FESS procedures, and also included patients who underwent FESS in combination with nasal surgery (septoplasty and / or turbinectomy). Both primary and revision cases were included. Patients were discharged on the day after surgery and were recommended to perform saline nasal irrigation more than 4 times a day. Information on patient characteristics, indications for surgery, extent of surgical intervention, as well as bleeding and adhesion was recorded.

[0085] (Surgical Categories) - Surgery was performed according to the minimally invasive sinus technique (MIST) (including combinations of maxillary antrostomy, ethmoidectomy, sphenoidotomy, frontal sinus (Draf IIa or Draf III), turbinectomy and / or septoplasty). FESS procedures were classified into the following two groups: complete FESS (including all sinus surgeries - maxillary antrostomy, ethmoidectomy, sphenoidotomy, and frontal sinusotomy) or limited FESS (all those that did not meet the criteria for complete FESS, usually including antrostomy and ethmoidectomy). The extent of surgery was defined by the patient's condition and the surgeon's clinical judgment.

[0086] (Materials Used) - PuraSinus (3D-Matrix, approved as PuraStat in Australia) / RADA16-I (SEQ ID NO: 1) at 2.5% w / v in water is a clear SAP solution supplied sterile in ready-to-use prefilled syringes (available in 3 mL and 5 mL formats) with a thin application nozzle suitable for endoscopic catheter administration. The syringes are stored between 2 °C and 8 °C. Once the FESS procedure was completed, PuraSinus was applied to the surgical wound in a thin, flat layer as close as possible to the bleeding point. A similar application technique has been previously outlined (M. Lee et al., Int. J. Surg. Case Rep. 41:461 - 4 (2017)). Gravity was sometimes used to reach the posterior part of the inferior turbinate. The tissue treated with this hydrogel could be seen via endoscopy considering the transparency of the gel.

[0087] (Outcomes and Objectives) - The objectives of this study were to evaluate the effectiveness of PuraSinus in achieving rapid intraoperative hemostasis, as well as to evaluate the incidence of delayed bleeding (more than 24 hours postoperatively) and adhesion formation by endoscopic observation of the nasal mucosa during follow-up visits. Primary bleeding was defined as bleeding occurring within 24 hours of the surgery.

[0088] (Data Analysis) - Statistical analysis was performed using descriptive statistics for continuous variables, and patient demographic information was recorded. The rates of successful hemostasis and the incidence of adhesion formation were calculated as a proportion of all patients who underwent FESS.

[0089] (Detailed Results) - The results were analyzed from a total of 94 human patients who underwent sinus surgery from May 2017 to February 2021. Thirty-eight males and 56 females, aged in the range of 18 to 83 years (median 48 years, mean ± standard deviation (SD) 47.9 ± 15.8 years), were included in this study. The patient characteristics are summarized in Table 4. The most common indication for surgery was chronic rhinosinusitis (CRS) with or without nasal polyposis. Twenty-eight patients underwent a complete FESS procedure, and the majority of them also underwent septoplasty (25 / 28) and turbinate reduction surgery (27 / 28). There were 66 patients who underwent a limited FESS procedure, and the majority of them also underwent septoplasty (59 / 66) and turbinate reduction surgery (62 / 66). Among the total of 94 patients, 7 were considered revision surgeries. The design of these flowcharts is shown in FIGS. 10A and 10B.

Table 4-1

Table 4-2

[0090] (Postoperative bleeding) - A total of six patients (6.4%) experienced postoperative bleeding. These results are summarized in Table 5. One of the six patients had undergone total FESS, and the other five had undergone limited FESS. Three patients had postoperative bleeding within 24 hours after surgery. One of them may have had an existing bleeding disorder. All three of these patients required cauterization of the surgical wound for hemostasis. In addition to cauterization, Surgiflo (absorbable gel hemostatic agent) was used in one patient, and a second application of PuraSinus was successfully used in another patient. Of the three patients who experienced delayed bleeding (i.e., more than 24 hours after surgery), only one, who had previously been diagnosed with a bleeding disorder, required intervention. The other two patients with secondary bleeding after surgery were managed conservatively. Overall, four patients (three with primary bleeding and one with secondary bleeding), all of whom had undergone septoplasty and turbinectomy in combination with their limited FESS (three patients) or total FESS (one patient), required intervention for bleeding (4 / 94: 4.3%). Thus, the application of PuraSinus (RADA16-I at 2.5% in water; SEQ ID NO: 1) in achieving hemostasis in this study was 95.7% (90 / 94). No rebleeding was reported from either patients with primary bleeding or patients with secondary bleeding.

Table 5

[0091] (Adhesion) - The nasal endoscopy of each patient was performed at the first follow-up visit between 7 and 30 days after surgery (median 15 days, mean ± standard deviation (SD) 14.0 ± 3.7 days). The subsequent follow-up examinations of each patient were performed on average on the 42nd day within the range of 28 to 79 days after surgery. No residual PuraSinus (SEQ ID NO: 1) hydrogel was observed, and all patients reported being comfortable and having no pain at the surgical site. A total of 23 patients (24.5%) were found to have adhesion formation during the entire follow-up period, of which 8 had undergone total FESS and 15 had undergone limited FESS. Only 18 of these 23 patients were found to have adhesions at the first follow-up visit. The most common site where adhesion formation was found was the inferior turbinate / septum (n = 11 / 23). Table 6 summarizes the location and characteristics of adhesions found in patients who underwent FESS. Debridement of the observed adhesions was performed by either suction (n = 13) or forceps (n = 10) in the outpatient setting in these cases. Subsequently, at the second follow-up examination, a total of 84 patients were found to have no adhesions (n = 71) or to have adhesions that were easily removed by suction division (n = 13). Therefore, the desirable outcome of the intraoperative procedure with PuraSinus (SEQ ID NO: 1) in achieving recovery without adhesion formation in these patients was approximately 90% (n = 84 / 94). None of the patients who required additional intervention for hemostasis subsequently showed adhesion formation. Furthermore, none of the patients in this case series required revision surgery.

Table 6

[0092] (Investigation) - The inventors have shown that the application of the peptide hydrogel PuraSinus (RADA16-I; (SEQ ID NO: 1)) is effective in limiting bleeding and adhesions while avoiding nasal packing after FESS in most patients in this study. Up to 25% of the postoperative bleeding in FESS can occur within 24 hours. Significant bleeding can occur up to 6 weeks postoperatively, but the most common time frame is between 1 and 2 weeks after FESS (Pant H., Otolaryngologic Clinics of North America, 2016; 49(3): 655-76). Similarly, in the inventors' study, of the 6 patients who experienced bleeding, 3 had postoperative bleeding on the 2nd, 5th, and 9th days. The other half of the patients recorded primary bleeding (occurring within 24 hours). This discrepancy may be due to the smaller number of the inventors' cohort, and contributing to this was the fact that 1 of the patients with primary bleeding had a suspicion of a blood coagulation disorder. Unfortunately, there are variations in the definitions in the literature when investigating bleeding during and after FESS. For example, some studies have identified postoperative bleeding in FESS as severe bleeding (the patient needs to return to surgery for transfusion or hemostasis), or recorded bleeding as a complication only when bleeding requires readmission (S. Suzuki et al., The Laryngoscope. 2015; 125(8): 1785-91; V. Siedek et al., European Archives of Oto-Rhino-Laryngology, 2013; 270(1): 141-8; Y-P. Wang et al., Journal of the Chinese Medical Association, 2011; 74(1): 16-21; H. Khoury et al., The Laryngoscope, 2021; 131(5): E1422-E8). Excessive perioperative bleeding occurs in approximately 5% of patients after FESS, and 0.8% of massive bleeding requires transfusion (C. Hopkins et al., The Laryngoscope, 2006; 116(8): 1494-9; J.L. Antisdel et al., The Laryngoscope, 2016; 126: S5-S13).To the same extent, in the study by the present inventors, the rate of patients with any bleeding was 6.4%, but the rate of patients who required additional treatment was only 4.2% (n = 4 / 94). None of the patients in this study required blood transfusion. In a large retrospective study of 3,402 patients over 25 years, Stankiewicz et al. (2011) only regarded bleeding as a complication when packing or surgery was required for bleeding control (JA Stankiewicz et al., The Laryngoscope, 2011; 121(12): 2684 - 701). Therefore, according to the definition of the present inventors regarding hemostatic effectiveness, similar to the definition of Stankiewicz et al., 95.7% (n = 90 / 94) of the patients who underwent FESS had no bleeding postoperatively, or the patients with bleeding did not require any intervention.

[0093] The rates in the study by the present inventors were similar and were within the lower range of the previously reported incidence rates (R. Valentine et al., American Journal of Rhinology & Allergy, 2010; 24(1): 70 - 5; JL Antisdel et al., American Journal of Rhinology & Allergy, 2011; 25(4): 268 - 71). It was found that a total of 24.5% of the patients had adhesions throughout their postoperative follow - up appointments. However, only 10 of these required sharp debridement, while the remaining 13 were easily separated by suction debridement. As a result, in this study, 89.4% (n = 84 / 94) of the patients had no adhesions at the second follow - up examination. In that case, 71 patients had no adhesions detected during the first follow - up period, and 13 of these patients had the adhesions easily removed. It was found that the majority of the patients with adhesions were resolved after the intervention. None of the patients in this study required revision surgery.

[0094] The incidence of adhesion formation and bleeding after paranasal sinus surgery varies considerably in the literature and may be related to the heterogeneity of the studies, with variables that confound including the extent of surgery, septoplasty or other adjunctive procedures involved, patient factors (smoking and co-existing diseases), and the type of nasal packing used. Wang et al. (2015) conducted a systematic review and meta-analysis to compare and contrast the effectiveness of non-absorbable nasal packing with that of absorbable nasal packing in FESS (T-C. Wang et al., European Archives of Oto-Rhino-Laryngology, 2015;272(8):1825-31). Their study found that there may be evidence that absorbable nasal packing may provide some improved outcomes compared with non-absorbable packing after FESS, but there was a lack of homogeneity between the studies (ibid.). They were unable to draw definitive conclusions from their review and suggested the need for more randomized clinical trials on the topic. Similar findings have been demonstrated in another meta-analysis and systematic review regarding middle meatus packing after FESS, where Hobson et al. (2015) concluded that it does not significantly reduce the risk of adhesion formation (CE. Hobson et al., American Journal of Rhinology & Allergy, 2015;29(2):135-40).

[0095] PuraSinus at 2.5% w / v in water as a topical hemostatic agent has been widely used in gastrointestinal endoscopic surgery (G. de Nucci et al., Endoscopy, 2020; 52(09): 773 - 9; M. Pioche et al., Endoscopy International Open. 2016; 4(4): E415; S. Subramaniam et al., Endoscopy, 2021; 53(01): 27 - 35; S. Subramaniam et al., United European Gastroenterology Journal, 2019; 7(1): 155 - 62; T. Uraoka et al., Gastrointestinal Endoscopy, 2016; 83(6): 1259 - 64), as well as in cardiovascular surgery (S. Giritharan et al., Journal of Cardiothoracic Surgery, 2018; 13(1): 1 - 7; H. Masuhara et al., Annals of Thoracic and Cardiovascular Surgery, 2012: oa.12.01977; M. Morshuis et al., The Journal of Heart and Lung Transplantation, 2019; 38(4): S194), with a hemostasis success rate in the range of 72.6% - 100% (S. Sankar et al., Frontiers in Bioengineering and Biotechnology, 2021; 9: 465). However, clinical evidence within the field of ENT surgery is limited. Lee et al. (2017) published a case series of 60 patients who underwent turbinectomy / resection (TRS / turbinectomy). Comparable to this study, the exclusion criteria included any concomitant hemostatic adjuvant, and similar techniques of PuraSinus (referred to as PuraStat in that paper) application and standard postoperative instructions were used. Lee et al., (2017) found that PuraSinus was highly effective, and none of their patients experienced postoperative bleeding or adhesion formation.Despite this excellent outcome, it differs from the overall adhesion rate and bleeding rate (24.5% and 6.4% respectively) in the inventors' study. This may be due to several differences between these two studies. The case series by Lee et al. included patients who underwent only TRS / turbinateplasty, while this study tested a cohort of patients who underwent (complete or limited) FESS, including TRS, septoplasty, and polypectomy. Since this includes a wider range of diseases and surgical interventions, there may have been an increased risk of adhesions and bleeding (Stankiewicz JA. et al., The Laryngoscope, 2011;121(12):2684 - 701). Furthermore, the data was recorded only from the first follow-up at 4 weeks in a case series of 60 patients, which differs from this study where the follow-up was longer up to several months and led to the identification of an additional 5 patients with adhesions (however, only 18 out of 23 patients were identified at the time of the first follow-up booking). In the case series by Lee et al. (2017), all patients were described as having complied with their postoperative care and nasal irrigation. Saline nasal irrigation is widely recommended after FESS and plays an important role in wound healing as well as in preventing crust formation and adhesion formation (Lee et al., Archives of Otolaryngology-Head & Neck Surgery, 2007;133(8):776 - 9; Shoman et al., Journal of Otolaryngology-Head & Neck Surgery=Le Journal d’oto-rhino-laryngologie et de chirurgie cervico-faciale, 2009;38(1):112 - 8; Yoo F. et al., International Forum of Allergy & Rhinology, 2018;8(1):32 - 40). Patient compliance with postoperative care was not formally recorded in the inventors' study and could have affected the patients' outcomes.Despite these differences, the inventors found promising overall efficacy of PuraSinus for hemostasis (95.7%) and for prevention of adhesion formation (89.4%).

[0096] This study had several limitations. Due to the absence of a control group, it was difficult to compare with other types of hemostatic agents or packing. All surgeries were performed by a single surgeon at a single facility, but the patient population was heterogeneous (including disease and degree of surgery, surgical indications, and additional surgeries performed). Also, specific variables that could affect the outcome (such as concomitant co-morbidities, smoking history, and patient compliance) were not recorded (as noted above). Furthermore, it would have been useful to identify the site of postoperative bleeding as was done for the site of postoperative adhesions. Quantitative information for evaluating the outcome could be obtained, for example, by using a severity grading using the scarring component of the Lund-Kennedy endoscopic score (OA Henriquez et al., The Laryngoscope, 2013;123(11):2615 - 9).

[0097] However, these results showed that the relatively simple technique of applying PuraSinus in patients undergoing sinus surgery was effective in achieving hemostasis, reducing adhesion formation, and avoiding nasal packing in most patients.

[0098] Although the present invention has been particularly shown and described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made in those embodiments without departing from the scope of the invention as included by the appended claims.

Table 7 - 1

Table 7 - 2

Table 8 - 1

Table 8-2

Table 9-1

Table 9-2

Claims

1. A composition for use in reducing negative complications from endoscopic turbinate plasty or functional endoscopic sinus surgery (FESS) in subjects, wherein the use is This involves applying an effective amount of one or more self-assembling peptides (SAPs) in a solution, The peptide is selected from a) SEQ ID NO: 1 (RADA16), having a purity of at least 65% by weight / vol. and / or a concentration between 0.5% by weight / vol. and 5% by weight / vol. in water or physiological buffer, and b) SEQ ID NO: 3 (IEIK13), having a major peptide purity of at least 70% by weight / vol. and a total peptide concentration between 0.05% by weight / vol. and 5% by weight / vol. in water or physiological buffer, wherein the application is performed during or immediately after endoscopic turbinoplasty or FESS, and the SAP solution is applied to the surgically treated area in the turbinates through a catheter, thereby forming a clear, absorbable, adhesive gel that comes into contact with the wound, thereby reducing negative complications from the endoscopic turbinoplasty or FESS of the subject.

2. The composition for use according to claim 1, wherein the SAP solution is applied to one or more sinus orifices, thereby preventing the formation of “blockages” or obstructive adhesions that could close off air circulation in the sinuses and create an environment for chronic sinus infection.

3. The composition for use according to claim 1, wherein even in cases where postoperative adhesions occur, the condition of the subject is resolved without trauma.

4. The composition for use according to claim 1, wherein the subject is a human.

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