Compositions and methods for wound healing and repair of damaged nerves

Abstract

Aspects of embodiments of the present disclosure relate to compositions and methods for treating wounds and / or promoting wound healing, as well as for regenerating injured mammalian nerves. Additionally, the present invention relates to the application of a therapeutically effective amount of an endothelin-B receptor agonist. In particular, the present application relates to a method for treating skin or cutaneous wounds and injured cranial or peripheral nerves using IRL-1620 (sovateltide). The composition may include an endothelin-B receptor agonist, including a peptide such as sovateltide (IRL-1620). Delivery systems are also described, including gels, sponges, gauze, and meshes incorporating the drug for topical application.

Description

[Technical Field]

[0001] The present disclosure relates to methods and compositions for promoting the rate and quality of wound healing, including regeneration of neurovascular and muscular tissue in individuals in need thereof. The methods include administering an endothelin B (ETB) receptor agonist together with one or more bioactive factors or stem cells. [Background technology]

[0002] Wound healing is a complex process involving multiple stages. These include (1) coagulation, which begins immediately after injury; (2) inflammation, which begins minutes later; (3) the migration and proliferation process (granulation phase), which begins within hours to days; and (4) the remodeling process, which results in the development of full-strength skin [1-3]. Coagulation controls hemostasis and initiates healing by releasing various growth factors and cytokines from degranulated platelets. During the inflammatory phase, platelet aggregation and coagulation form a matrix that traps plasma proteins and blood cells and induces the influx of various cell types. Neutrophils are the first cells to arrive and are responsible for phagocytosis of contaminating bacteria, digestion of fibrin clots, release of mediators that attract macrophages, and activation of fibroblasts and keratinocytes [3]. Macrophages secrete cytokines / growth factors (e.g., interleukin-1 (I-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF)) that digest pathogens, debride the wound, and stimulate fibroblasts and endothelial cells. Overall, the inflammatory phase is important for fighting infection and promoting the migration and proliferation phase of wound healing.

[0003] There are two types of wounds: acute and chronic. Acute wounds heal completely within 4–6 weeks. When the healing process persists for more than 6 weeks, they become chronic wounds. Chronic wounds, such as venous, diabetic, or pressure ulcers, are one of the most significant unmet medical needs in the world today and are a significant complication of diabetes, resulting in significant morbidity, lost productivity, and increased healthcare costs. Diabetic foot ulcers are a significant cause of morbidity and the most common reason for hospitalization in diabetic patients. Approximately 15% of diabetic patients will develop a chronic ulcer during their lifetime. Of these, foot ulcers precede 70–90% of those requiring lower-limb amputation.[4]

[0004] Diabetic wounds exhibit elevated levels of matrix metalloproteinases (MMPs), increased proteolytic degradation of ECM components, and growth factor inactivation, ultimately resulting in an abnormal ECM that cannot support healing [5,6]. Abnormal nitric oxide (NO) production also contributes to the pathogenesis of impaired healing. Cells such as keratinocytes, fibroblasts, and macrophages are dysfunctional in their expression and response to many growth factors and cytokines. Therefore, these wounds typically do not respond to most treatments. For these reasons, proactive strategies to restore the abnormal extracellular microenvironment of diabetic wounds to more closely resemble normal ECM components in many aspects may be most advantageous. Regulatory pathways consist of a complex network, making the compensatory adjustments necessary for wound repair challenging. Therefore, proactive healing strategies that replace missing or dysfunctional extracellular matrix (ECM) components may be most advantageous.

[0005] (Prior Art Background)

[0006] Over the past decade, vascular endothelial growth factor (VEGF) has been extensively studied and documented for its multifaceted functions in the central nervous system (CNS) and its supporting physiological environment. In addition to its well-known functions, such as angiogenesis, enhanced vascular permeability, and glial proliferation, VEGF is also involved in more recently recognized functions, such as neuroprotection and neurogenesis. Its neurogenesis function in particular has attracted considerable attention in recent years, and several research groups are working to elucidate this activity. Along with this trend, our knowledge of VEGF receptors has also increased, and although some suggestions regarding neuroprotective mechanisms have emerged through ongoing research, researchers have often only been able to address a small portion of VEGF signaling. Along with flt-1 (VEGF receptor 1) and flk-1 (VEGF receptor 2), neuropilin (NP) has frequently been described as being involved in the neuroprotective effects of VEGF. The direct and indirect neuroprotective effects of VEGF, various signaling pathways, and neurogenesis mediated by this factor are discussed in the context of new insights into the biological mechanisms of VEGF and closely related and interacting molecules [7].

[0007] Leukocytes, particularly neutrophils and macrophages, remain in the surrounding tissue and secrete various proteases, including matrix metalloproteinases (MMPs) and serine proteases. [8] Overaccumulation of these enzymes impedes matrix remodeling. [9] Protease inhibitors are thought to be beneficial for wound healing.

[10] Another characteristic of some chronic wounds is reduced or absent vascularization, which impedes the supply of nutrients to newly formed tissue.

[11] Existing techniques for promoting chronic wound healing are initially managed by debridement of necrotic tissue, antibiotic treatment when appropriate, and regular dressing application. [2] Other dressings that may be used include hydrogels, hydrocolloids, and alginates. Venous ulcers are treated with compression therapy, while arterial or diabetic ulcers require regular dressing changes. Pressure ulcer healing is promoted by removing pressure from the injury site. Other physical devices such as laser therapy, hyperbaric oxygen, and electrical stimulation for arterial ulcers have also been used to promote wound healing [2,12,13].

[0008] The use of tissue-engineered skin, such as Dermagraft or Apligraf, is an option for wounds that do not respond to these interventions. This treatment prevents bacterial infection and allows wound healing via normal repair processes [14,15]. The promotion of wound healing with such skin substitutes presupposes the presence of a vascular supply at the existing wound site. Another approach to wound healing involves the administration of growth factors / cytokines, which have been shown to promote cell proliferation in vitro and accelerate wound healing in some animal models. These include IL-1, platelet-derived growth factor (PDGF), EGF, VEGF, TGF-β, and bFGF [2]. Procuren (Curative Technologies) is an autologous platelet release product containing at least five growth factors that support granulation tissue formation and re-epithelialization. This autologous growth factor mixture has shown some success in human subjects with ulcerative limb lesions

[16] . However, overall, the results of many clinical trials using growth factors / cytokines have been disappointing. For example, EGF failed to heal venous stasis ulcers, and IL-1 was ineffective in treating pressure ulcers. [2] Similar results have been reported with bFGF.

[17] The reasons for the lack of efficacy are unclear, but multifactorial effects of growth factors / cytokines that are unfavorable for healing may be involved.

[0009] Jackson's patent

[18] uses activated protein C (APC) to aid in wound healing. According to the patent, APC has been reported to have anti-inflammatory properties and to directly activate the protease gelatinase A [18, 19].

[0010] Various cell types, including smooth muscle cells, fibroblasts, and endothelial cells, secrete gelatinase A. Gelatinase A degrades basement membrane collagen

[20] , allowing cells to invade the interstitium, playing an important role in physiological remodeling and angiogenesis

[21] . Furthermore, APC promotes endothelial cell regeneration after wounding in vitro, stimulates re-epithelialization, fibroblast invasion, and angiogenesis in chick embryos, and promotes wound healing in a rat wound model. Together with the anticoagulant, anti-inflammatory, and gelatinase A-activating functions described above, these functions strongly suggest that APC, its functional fragments, and its precursor (i.e., protein C) may be useful in treating wounds, especially those with slow healing.

[0011] Another patent

[22] describes a topical composition for treating skin wounds using whole colostrum. Growth factors, immune factors, enzymes, and micronutrients in colostrum are physiologically active and contribute to wound healing. The growth factors described include epithelial, fibroblast, insulin-like I and II, transforming alpha and beta, and platelet-derived nerve growth factor. Immune factors include immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, and lactalbumin. Enzymes include lysozyme, peroxidase, protease, and xanthine oxidase. Micronutrients include carbohydrates, amino acids, vitamins, and minerals.

[0012] Human plasma fibronectin is described in a patent

[23] . This patent provides a formulation that allows for sustained release of fibronectin at the wound site and extended contact time for effective absorption. Fibronectin is involved in all stages of wound healing. The biological activities of fibronectin related to wound healing include cell recruitment, opsonization of particulate debris, and promotion of wound contraction

[24] . Furthermore, topical fibronectin has been shown to be useful in increasing the rate of wound healing, such as in corneal wounds [25, 26] and leg ulcers

[27] . The patent focuses on gel and cream formulations.

[0013] Patent

[28] describes the topical or transdermal delivery of a wound healing promoting composition containing peptides such as nitric oxide and / or thyrotropin releasing hormone (TRH), and / or GnRH (gonadotropin releasing hormone).

[0014] The growth factor approved by the U.S. Food and Drug Administration (FDA) for the treatment of diabetic foot ulcers is recombinant PDGF-BB (becaplumin), available as a topical cream. PDGF-B is known to be a potent growth factor and chemotactic factor for stromal cells and can increase wound vascularization by stimulating angiogenesis.

[0015] A US patent

[29] supports wound repair using angiopoietin-like protein 4 (ANGPTL4). ANGPTL4 has been shown to play a context-dependent role in angiogenesis and vascular permeability [30-32]. ANGPTL4 deficiency in mice resulted in delayed wound re-epithelialization, reduced matrix protein expression, increased inflammation, and impaired wound-associated angiogenesis [33, 34].

[0016] Specific areas of wound healing include neuroprotection and regeneration of injured axons and nerve tissue. When axons in the mammalian peripheral nervous system (PNS) are severely injured (i.e., by compression or transection), several events can occur. First, if left unmodified, the distal stump almost always degenerates (Wallerian degeneration). This degeneration is accompanied by simultaneous chromatolytic changes in the proximal perikarya. If nerve injury is sufficiently severe, proximal axons also degenerate, followed by perikalion degeneration and (usually) death. If nerve injury is less severe and unmodified, several biochemical changes are initiated in the remaining proximal segment. These proximal changes involve a complex series of cell body responses to injury and are thought to prepare the intact segment for regeneration. These include changes in axonal transport properties, protein processing, and nucleic acid synthesis. Morphologically, regenerative growth cones often emerge from the proximal stump, and axons begin to regrow from the proximal stump toward their original target region.

[0017] Such injuries likely produce lateral sprouts from adjacent axons that did not undergo Wallerian degeneration. Newly growing neurites likely guide the denervated distal segment toward the denervated target area. Reactive Schwann cells presumably provide a vehicle for regeneration to the target by supplying diffusible growth-promoting factors and providing an appropriate growth surface established by the plasma membrane and / or basement membrane or extracellular matrix. These morphological and biochemical changes depend primarily on several factors, including the severity of the injury and its proximity to the perikarya, the size of the injured axon, and the species involved. For example, higher vertebrates have a lower capacity for effective regeneration of peripheral nervous system (PNS) axons.

[0018] In the central nervous system (CNS), spontaneous attempts at regeneration are often rapidly aborted, resulting in a completely degenerated proximal stump and perikarya.

[0019] Trophic factors from nearby tissues may have a higher trophic potential than the target tissue. Consequently, the length of the injury gap has been shown in the prior art to be a major factor determining the success of functional regeneration. This lack of target specificity has been suggested in the literature as the underlying cause of frequent neuromas and inadequate contact with other tissues. Accordingly, significant improvement has been observed after nerve anastomosis has been performed. The current method of choice for neurosurgical repair of peripheral nerve injuries is simple anastomosis of the severed nerve ends.

[0020] Many studies have been conducted to evaluate the regeneration of nerves and / or axons. Axons resulting from traumatic injury often fail to regenerate spontaneously in the adult central nervous system. Injury to the peripheral nervous system can lead to neuropathy, resulting in muscle weakness or paralysis, reduced or lost sensation, unpleasant and painful neuropathy, and autonomic dysfunction.

[35] The peripheral nervous system has some regenerative capacity.

[0021] (Background of existing compositions and formulations)

[0022] The gel formulations comprise a water-soluble, pharmaceutically acceptable polymer formulated with an effective amount of fibronectin. Examples of such compounds include vinyl polymers, such as polyacrylic acid, polyoxyethylene-polyoxypropylene block copolymers, such as poloxamers, and cellulose derivatives, such as hydroxypropyl cellulose (HPC). The polymer provides a viscosity of 50,000 to 1,000,000 cps at room temperature. The cream formulations are prepared from a commercially available cream base, i.e., Schering base (Schering Canada Inc., Point-Claire, Quebec), and have a viscosity of 60,000 to 80,000 cps at room temperature. The sustained-release system provided by the gel formulations of the present invention provides sustained release of fibronectin to the wound site. This characteristic of these formulations allows for less frequent application to the wound, resulting in less disruption to the healing process. Such formulations maintain fibronectin delivery for up to 24 hours. A preferred embodiment is a twice-daily treatment schedule, based on kinetic data.

[0023] The patent

[36] describes a wound healing composition consisting of an aqueous mineral oil emulsion that is easily spreadable and forms a stable, moist layer when applied to moist skin or mucosal surfaces.

[0024] The composition may take the form of a gel, cream, lotion, ointment, solution, solid, adhesive patch with a reservoir (allowing for slow release of the composition), etc., and may be rubbed or sprayed onto the affected area, including the skin, subcutaneous tissue, muscle, fascia, blood vessels, and nerves, in need of regeneration or healing.

[0025] The goals of wound repair include accelerating wound closure with minimal scar formation.

[0026] Several studies have examined the effectiveness of epidermal growth factor (EGF) in wound healing, with mixed results. Thornton et al. (1982) applied EGF topically to burns in rats

[37] . Only a non-significant healing effect was observed compared with the control group. Niall et al. (1982) reported that topical application of EGF to wounds in mice accelerated wound healing

[38] . Brown et al. (1986) applied EGF topically to wounds in miniature pigs

[39] . They reported an increased rate of epithelialization of split-thickness wounds in vivo. Buckley et al. (1985) examined the effects of sustained release of EGF from subcutaneously implanted sponges in rats

[40] . They concluded that the sustained local presence of EGF accelerated the wound healing process.

[0027] Studies by Lawrence et al. (1986) and Sporn et al. (1983) suggested that administration of transforming growth factor (TGF) accelerated wound healing in rats [41, 42], and Lawrence et al. (1986) observed an even more accelerating effect with the combination of TGF, EGF, and platelet-derived growth factor (PDGF)

[41] . Furthermore, Shultz et al. (1987) reported that topical application of transforming growth factor alpha (TGF-α) and vaccinia growth factor (VGF) in an antibiotic cream accelerated epithelial regeneration in partial-thickness skin burns (second-degree burns) on the backs of pigs

[43] .

[0028] In recent years, there has been growing interest in IGFs, such as insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II), also known as somatomedin C, as described in a patent

[44] . Froesch et al. (1985) reported that the primary effect of IGFs is on the growth and differentiation of mesodermally derived cells

[45] . Furthermore, they reported that IGFs, present in high concentrations in serum as bound forms, may fulfill several functions, including replacement of dead cells, repair mechanisms, matrix synthesis, and homeostatic stabilization of cells, possibly preventing transformation and dedifferentiation

[45] .

[0029] (Prior Art Background in Nerve Regeneration)

[0030] Simple anastomosis is insufficient when the damage and degeneration are extensive and the distance between the remaining proximal and distal stumps is excessive. An alternative approach is to use a structure or "bridge" that spans the gap from the proximal stump to the distal portion of the nerve or the target tissue itself. In animal studies, materials used to bridge the gap include peripheral nerve grafts, interstitial space, Millipore, and silicone tubing.

[0031] Of particular interest are artificially manufactured, commercially available "nerve cuffs" or "nerve guide tubes," which are implanted and extended between stumps. Nerve guide tubes are generally made of either silicone or biodegradable materials. Nerve guide tubes can be filled or coated with a matrix that promotes nerve growth, such as laminin, to promote nerve growth over longer distances than unmodified nerve guide tubes alone.

[0032] Want et al. demonstrated the beneficial effects of branched-chain amino acids on the neuronal survival and axonal regeneration of retinal ganglion cells via the mTOR (target of rapamycin) pathway

[46] . Nucleic acid therapy may improve regeneration by enhancing the intrinsic growth potential of neurons and overcoming environments that inhibit neurite outgrowth. These nucleic acids regulate gene expression by overexpressing nerve growth factors or silencing growth inhibitory molecules

[47] . Other studies have evaluated neuronal regeneration using the olfactory nerve. Dysfunction of this nerve causes loss of smell. In one study, the combined delivery of two growth factors, vascular endothelial growth factor and platelet-derived growth factor, increased the number of mature olfactory neurons

[48] . Furthermore, the neuroprotective activity of curcumin was investigated. The effects of local and sustained low-dose curcumin treatment on nerve regeneration after rat sciatic nerve crush injury were investigated. Curcumin treatment resulted in increased expression of compact myelin protein, increased myelin sheath thickness, and increased motor and sensory nerve conduction velocity. Furthermore, curcumin treatment reduced reactive oxygen species (ROS) production (prominently produced by macrophages), inhibited lipid peroxidation, and increased the expression of the transcription factor Nrf2. This antioxidant potential likely contributes to the beneficial effects of curcumin after SNC injury

[49] .

[0033] Advanced conduit design and manufacturing techniques have made it possible to create autograft-like structures within NGCs with great precision. To this end, strategies using biopolymers, cells, growth factors, and physical stimuli have been developed over the past few decades to develop a variety of NGCs, ranging from simple hollow tubes to complex conduits incorporating one or more guidance cues

[35] . One study demonstrated that the combination of nerve conduits and stem cells promoted recurrent laryngeal nerve recovery after injury. Results showed that the combination of laminin-chitosan-PLGA nerve conduits with Schwann cells and neural stem cells promoted nerve regeneration (P<0.05), and the effect was superior to that of autografts (P<0.05)

[50] .

[0034] Non-pharmacological agents have been used for nerve regeneration, including electrical stimulation. One study evaluated the regenerative effects of implanting electrodes with different contacts into a resected sciatic nerve. This method demonstrated an increase in the sciatic function index, an increase in the amplitude of the compound muscle action potential, an increase in motor nerve conduction velocity, and a decrease in muscle atrophy

[51] . In another study of peripheral nerve injury, the combination of electrical stimulation and neural crest stem cells significantly promoted nerve regeneration after injury and repair. Results were comparable to those of autologous transplantation

[52] .

[0035] After injury, axons can use several different strategies to reconnect with target tissue. First, regrowth must be initiated, which can begin at the tip of the severed axon end connected to the cell body, from branches extending from it, or from newly emerging axons originating from the cell body. To reach their target, axons can travel along the entire length of their original pathway or utilize different ectopic pathways. The necessary regrowth can be extensive; regenerating axons may instead form functional connections with nearby neurons or connect with newly generated neurons after cell migration, shortening their growth requirements. Peripheral nerve injuries involving large intersegmental gaps are typically repaired with autologous nerve grafts

[53] . An alternative approach, particularly in severe injuries, is end-to-side neurorhaphy, which joins the severed distal nerve stump to an adjacent, intact donor nerve trunk

[54] . This technique has been used for a variety of peripheral nerve injuries, but results have been mixed, and its use is limited by a lack of randomized clinical trials

[54] . Application of this approach with an endothelin B receptor agonist such as sovateltide (IRL-1620) may aid in functional recovery and extend the clinical utility of the neurorrhaphy approach.

[0036] Growth factors (GFs) (e.g., epithelial, fibroblast, insulin-like I & II, transforming O & B, platelet-derived, neural), immune factors (e.g., immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, lactalbumin), enzymes (e.g., lysozyme, peroxidase, proteases, xanthine oxidase), and micro- and macronutrients (e.g., carbohydrates, amino acids, vitamins, minerals) have also been proposed for wound healing and repair of injured nerves.

[0037] (Background of endothelin analogs related to wound healing)

[0038] Many compositions currently exist that enable the healing of tissue structures. Endothelin has been reported to promote numerous cascades that are effective in wound healing. A study by Qin D. et al. investigated the role of endothelin-1 (ET-1) in retinal pigment epithelial (RPE) cell proliferation, migration, and secretion of extracellular matrix (ECM) components such as type I collagen and fibronectin in vitro. The results showed that ET-1 promotes proliferation, migration, and ECM secretion via the protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) signaling pathways. This suggests that ET-1 may play an important role in the development of proliferative vitreoretinopathy (PVR). PVR represents an excessive wound healing response mediated by ECM molecules

[55] .

[0039] Endothelins have also been studied in liver wound healing. For example, a study by Khimji et al. (2011) found that ET-1 production is increased and that the cellular origin of ET-1 shifts from endothelial cells to astrocytes during liver injury. This creates a feedback loop that further enhances activation, astrocyte proliferation, and ECM protein production

[56] .

[0040] Another study by Lagares et al. (2010) demonstrated that endothelin-1 contributes to the effects of transforming growth factor B1 (TGFB1) on wound repair and skin fibrosis. TGFB1 induced ET-1 expression in human skin fibroblasts through Smad and activator protein 1 / JNK-dependent signaling. TGFB1's ability to induce fibrotic gene expression was dependent on ET-1. Adenovirus-mediated overexpression of TGFB1 and ET-1 in mouse skin was associated with accelerated wound closure, increased fibrosis, and increased scar formation

[57] . According to this study, ET-1 receptor antagonists, such as bosentan, may be a useful therapeutic tool for treating excessive scar formation and fibrosis-related diseases

[57] . Another study evaluated the role of ET-1 in the wound healing process. Skin wound healing was accelerated when ET-1 was not attenuated by bleomycin. ET-1 mice showed earlier granulation tissue formation and re-epithelialization

[58] .

[0041] Endothelins are involved in neurogenesis and neuroprotection. In one study, rats were treated with an endothelin B receptor agonist after ischemic stroke, compared with a no-drug control. Endothelin B receptor agonist rats showed increased angiogenesis as measured by vascular endothelial growth factor (VEGF), better preservation of neuronal cell numbers, and increased expression of nerve growth factor (NGF) [59-62]. Summary of the Invention

[0042] Aspects of embodiments of the present disclosure include novel compositions comprising endothelin B agonists that promote wound healing, including superficial and deep tissue structures, including, but not limited to, epithelium, subcutaneous tissue, muscle tissue, fascia, bone, and neurovascular tissue. In particular, the present invention is directed to the use of these compositions in treating patients with acute or chronic wounds, including diabetic foot ulcers. Furthermore, the present invention is directed to compositions that support nerve tissue regeneration after acute or chronic nerve injury, such as nerve transection during surgery. In preferred embodiments, the endothelin B agonist may have a variety of compositions, including, but not limited to, bioactive factors, stem cells, proteins, elements that promote tissue recovery, and the like. Local drug delivery in preferred embodiments can be achieved by topical application, placement into deep tissues during surgery, or injection. Systemic administration may also be an option when tissue damage is extensive. These delivery systems include intravenous, intramuscular, and oral dosage forms.

[0043] These and other features, aspects, and advantages of the present invention will become more apparent with regard to the following description, appended claims, and accompanying drawings. [Brief explanation of the drawings]

[0044] [Figure 1] The chromatogram of IRL-1620 (retention time 5.093) is shown. [Figure 2] 1 shows images of the cross-linking stage in an alginate hydrogel film before freeze-drying. [Figure 3] 1 shows an image of a fully cross-linked alginate hydrogel film before freeze-drying. [Figure 4] 1 shows sterilization by UV irradiation in a laminar airflow. [Figure 5] 1 shows an image of a freeze-dried hydrogel alginate film. [Figure 6] FTIR of the hydrogel film. [Figure 7] FTIR of sodium alginate. [Figure 8] FTIR of IRL-1620 is shown. [Figure 9] SEM images of the surface of alginate hydrogel films containing (a) 1% sodium alginate, (b) 1.25% sodium alginate, (c) 1.5% sodium alginate, and (d) 2% sodium alginate are shown. [Figure 10] 1 shows the optical transparency of the hydrogel film. [Figure 11] 1 shows the in vitro release pattern of IRL-1620 from formulations through goat skin for up to 26 hours. [Figure 12] 1 shows the in vitro release pattern of IRL-1620 from formulations through goat skin for up to 5 hours. [Figure 13] 1 shows the cumulative release rate pattern of IRL-1620 from the formulation through goat skin. [Figure 14] The % quantitative values ​​of stability samples of the developed formulation are shown below under refrigerated (2-8°C) and frozen (-20°C) storage conditions. [Figure 15] The chromatogram of IRL-1620 (retention time 5.093) is shown. [Figure 16] Microscopic images of niosomes F2 and F3 are shown. [Figure 17] Figure 1 shows the encapsulation efficiency of various formulations. [Figure 18] FTIR spectrum of IRL-1620. [Figure 19] FTIR spectrum of excipient (soybean lecithin) is shown. [Figure 20] Figure 1 shows the FTIR spectrum of niosomes (F3). [Figure 21] 1 shows a particle size distribution graph. [Figure 22] Zeta potential distribution peaks are shown. [Figure 23] Niosome gel G-3 (used for in vitro release and rat wound healing studies) is shown. [Figure 24] 1 shows the in vitro release pattern of IRL-1620 from formulation G-3 through goat skin. [Figure 25] 1 shows the cumulative release rate pattern of IRL-1620 from formulation G-3 through goat skin. [Figure 26] % quantitation of stability samples of the development formulation at room temperature and refrigerated conditions. [Figure 27A] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27B] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27C] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27D] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27E] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27F] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27G] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 27H] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 28A] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28B] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28C] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28D] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28E] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28F] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28G] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 28H] The effect of different treatments on collagen deposition in each ablation model is shown. [Figure 29A] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29B] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29C] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29D] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29E] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29F] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29G] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 29H] The effect of different treatments on VEGF expression in each resection model is shown. [Figure 30A] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30B] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30C] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30D] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30E] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30F] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30G]The effect of different treatments on vWF expression in each ablation model is shown. [Figure 30H] The effect of different treatments on vWF expression in each ablation model is shown. [Figure 31] Figure 1 shows the effect of different treatments on wound healing in different groups. [Figure 32A] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32B] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32C] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32D] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32E] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32F] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32G] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 32H] The effects of different treatments on inflammation, angiogenesis, and fibroblast proliferation in each excision model are shown. [Figure 33A] 1 shows collagen deposition in different incision models. [Figure 33B] 1 shows collagen deposition in different incision models. [Figure 33C] 1 shows collagen deposition in different incision models. [Figure 33D] 1 shows collagen deposition in different incision models. [Figure 33E] 1 shows collagen deposition in different incision models. [Figure 33F]1 shows collagen deposition in different incision models. [Figure 33G] 1 shows collagen deposition in different incision models. [Figure 33H] 1 shows collagen deposition in different incision models. [Figure 34A] VEGF expression in different incision models is shown. [Figure 34B] VEGF expression in different incision models is shown. [Figure 34C] VEGF expression in different incision models is shown. [Figure 34D] VEGF expression in different incision models is shown. [Figure 34E] VEGF expression in different incision models is shown. [Figure 34F] VEGF expression in different incision models is shown. [Figure 34G] VEGF expression in different incision models is shown. [Figure 34H] VEGF expression in different incision models is shown. [Figure 35A] Figure 1 shows vWF in different incision models. [Figure 35B] Figure 1 shows vWF in different incision models. [Figure 35C] Figure 1 shows vWF in different incision models. [Figure 35D] Figure 1 shows vWF in different incision models. [Figure 35E] Figure 1 shows vWF in different incision models. [Figure 35F] Figure 1 shows vWF in different incision models. [Figure 35G] Figure 1 shows vWF in different incision models. [Figure 35H] Figure 1 shows vWF in different incision models. DETAILED DESCRIPTION OF THE INVENTION

[0045] A method for promoting wound healing in tissues, including superficial and deep tissue structures, including, but not limited to, epithelium, subcutaneous tissue, muscle tissue, fascia, bone, and neurovascular tissue, is disclosed. In particular, the present invention is directed to the use of this composition in treating patients with acute or chronic wounds, including diabetic foot ulcers. Furthermore, the present invention is directed to a composition that supports nerve tissue regeneration after acute or chronic nerve injury, such as nerve transection occurring during surgery. The endothelin B receptor is important in neuroprotection and nerve regeneration, and also promotes the physiological wound healing cascade. The present invention relates to tissue healing using endothelin B receptor analogs, such as N-succinyl-[Glu9,Ala11,15]endothelin 1 (sovateltide, IRL-1620), BQ-3020, [Ala1,3,11,15]-endothelin, sarafotoxin S6c, and endothelin 3. Sovateltide (IRL-1620) increased cerebral blood flow, promoted angiogenesis and neurogenesis, and inhibited apoptosis and mitochondrial fission. IRL-1620 induced the differentiation of neural progenitor cells into mature neurons. The anti-apoptotic, anti-inflammatory, and antioxidant activities of IRL-1620 limit cell death and promote the formation of new nerve cells (neurogenesis) and new blood vessels (angiogenesis), enhancing wound healing and nerve regeneration.

[0046] (definition)

[0047] As used herein, the term "amount sufficient to achieve" refers to an amount that allows for the intended effect, such as increasing the amount of fibroblasts in a wound bed or improving conduction through a nerve pathway. Such an amount can be determined by various quantitative methods known to those of skill in the art based on the intended effect. As used herein, the term "application" or "administration" refers to any means of introducing a particular agent, composition, or force into a particular area or tissue. "Administration" or "application" can be a single administration, continuous administration over a treatment period, or intermittent administration. Methods for determining the most effective administration means and dosage are known to those of skill in the art and vary depending on the composition used for treatment, the purpose of treatment, the cells being treated, and the subject. Single or multiple administrations can be performed at dosages and patterns selected by the treating physician. Appropriate dosage formulations and administration methods are known to those of skill in the art. The route of administration can also be determined; methods for determining the most effective administration route are known to those of skill in the art and vary depending on the composition used for treatment, the purpose of treatment, the subject's health condition or stage of disease progression, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, inhalation, injection, and topical application. Administration can be used for both industrial and therapeutic purposes. The term "biodegradable" is used herein to describe substances such as polymers, compositions, and formulations that are intended to degrade during use. Biodegradable substances can be "biocompatible," meaning they are not harmful to living tissue. Non-limiting examples of biodegradable substances include poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) (optionally PEGylated).

[0048] The term "nerve tissue" refers to tissue that improves signal conduction in motor and sensory nerve pathways. It may include neurons, nerve cells, glial cells, axons, astrocytes, microglial cells, ependymal cells, oligodendrocytes, enteric glia, satellite cells, and Schwann cells. Nerve tissue also includes the central nervous system, including the brain and spinal cord, and parts of the peripheral nervous system that regulate and control the body's functions and activities.

[0049] As used herein, the term "tissue" refers to tissue of a living or dead organism, or any tissue obtained from or designed to mimic a living or dead organism.

[0050] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a desired effect. In therapeutic applications, the effective amount depends on the type and severity of the condition at issue and on individual subject characteristics, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. Those of skill in the art can determine the appropriate amount based on these and other factors. For in vitro applications, in some embodiments, the effective amount may depend on the size and nature of the intended application. It also depends on the nature and sensitivity of the in vitro target and the method of use. Those of skill in the art can determine the effective amount based on these and other considerations. An effective amount may comprise one or more administrations of the composition, depending on the embodiment. The dosage range for N-succinyl-[Glu9,Ala11,15]endothelin-1 (IRL-1620, sovateltide) is 0.00001 to about 1 mg, and may be administered once or multiple times daily, over several days, weeks, or months.

[0051] As used herein, the term "treatment" or "treating" includes preventing the onset of a disease, disorder, or condition in a subject susceptible to or having the disease, disorder, or condition, inhibiting the disease, disorder, or condition, e.g., arresting its progression, and alleviating or reversing the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition can also include ameliorating at least one symptom of a particular disease or condition.

[0052] (composition)

[0053] Aspects of the present disclosure relate to compositions or agents for regenerating lost or damaged tissue. The base composition comprises an endothelin B receptor agonist with or without stem cells. In some embodiments, the base composition may not include stem cells.

[0054] In some embodiments, the base composition may be combined with growth factors and / or cytokines known to promote tissue regeneration. Neuroregenerative compounds include, but are not limited to, growth factors, osmotic agents such as glycerol, diuretics such as furosemide or ethacrynic acid, steroids, transplanted stem cells, ferulic acid, arctigenin, espin isoform 1 polypeptide, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, nerve growth factor, vascular endothelial growth factor, ciliary neurotrophic factor, insulin-like growth factor, netrin-1, thyroid hormone, hepatocyte growth factor, luteolin, neuropilin, epidermal growth factor, transforming growth factor-β, human plasma fibronectin, platelet-derived growth factor, and basic fibroblast growth factor. Additionally, embodiments may include Procuren (Curative Technologies), an autologous platelet-release solution mixture containing at least five growth factors that support granulation tissue formation and re-epithelialization. This autologous growth factor mixture has shown some success in human subjects with ulcerative extremity lesions.

[0055] In some embodiments, the base composition may be combined with chronic wound healing strategies, including but not limited to debridement techniques, antibiotic treatment, compression therapy, laser treatment, hyperbaric oxygen therapy, and electrical stimulation. Such wound dressings may include hydrogels, hydrocolloids, or alginates and may be part of the embodiments.

[0056] In some embodiments, the base composition may be combined with compounds known to aid in wound healing, including activated protein C, which promotes endothelial cell regeneration. Other compounds include colostrum, which contains growth factors, immune factors, enzymes, and micro- and macronutrients present in colostrum that are bioactive and related to wound healing. Angiopoietin-like protein 4 (ANGPTL4) is another compound that aids in wound repair. ANGPTL4 has been shown to play a context-dependent role in angiogenesis and vascular permeability.

[0057] In some embodiments, the base composition may be combined with compounds that are shown to promote neuronal survival, recovery, and regeneration, including branched chain amino acids, nucleic acids that overexpress nerve growth factor, and curcumin.

[0058] In some embodiments, the base composition may be combined with immune factors, including immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, and lactalbumin.

[0059] In some embodiments, the base composition may be combined with hormones, including thyrotropin-releasing hormone and gonadotropin-releasing hormone.

[0060] In some embodiments, the basal composition may be combined with enzymes and / or micro- and macronutrients, including lysozyme, peroxidase, protease, xanthine oxidase, carbohydrates, amino acids, vitamins, and minerals.

[0061] In some embodiments, the base composition may be combined with a compound, such as gelatinase A, that has a degradative process that promotes wound healing.

[0062] In some embodiments, the stem cells used in the basic composition can include, but are not limited to, hematopoietic stem cells, embryonic stem cells, and platelet-rich plasma-derived stem cells.In some embodiments, stem cells can also be genetically modified as vectors that produce factors that promote the regeneration of damaged tissue, including nerve tissue.Finally, in some embodiments, stem cells can be transplanted into damaged tissue before administering endothelin B receptor agonist.

[0063] In some embodiments, the base composition may be combined with tissue-engineered skin such as Dermagraft or Apligraf. This treatment offers the opportunity to prevent bacterial infection and allow the wound to heal through normal repair processes.

[0064] (Preparation of Sovateltide (IRL-1620) Stock Solution in a Preferred Embodiment)

[0065] (drug substance)

[0066] IRL-1620 (98.5% purity) is manufactured and sold by American Peptide Company, Inc. (1271 Avenida Chelsea Vista, CA 92081, USA). Sodium alginate, glycerol, calcium chloride, and all other excipients and solvents were purchased from Merck.

[0067] (Drug Analysis)

[0068] (Analysis method for IRL-1620 (Sovateltide))

[0069] IRL-1620 was analyzed using an HPLC method. Analysis was performed by gradient HPLC using 1% TFA in water and 0.1% TFA in acetonitrile (55:45) as the mobile phase. The mobile phase was prepared fresh daily. The mobile phase was filtered through a 0.2 mm membrane filter to remove any particulate matter, mixed, and degassed by sonication before use. It was confirmed to be stable, with no precipitation over time or temperature. The absorbance of IRL-1620 was fixed at 215 nm without any interference. The detector sensitivity was set to 0.01 AUFS. Before injecting the solution, the column was equilibrated for at least 60 minutes with the mobile phase flowing through the system. Each solution was injected in triplicate, and the relative standard deviation (RSD) based on peak area was expected to be less than 1.0%. Detailed chromatographic conditions are as follows:

[0070] [Table 1]

[0071] (Preparation of IRL-1620 stock solution)

[0072] 1.0 mg of IRL-1620 lyophilized powder was accurately weighed and transferred to a 10 mL volumetric flask. The volume was adjusted with HPLC water to a concentration of 100 μg / mL IRL-1620 (IRL-1620 Stock-I). Next, a 100 μL aliquot of the above solution (Stock-I) was transferred to a 10 mL volumetric flask and the volume was adjusted with diluent to a concentration of 1.0 μg / mL. This solution was designated IRL-1620 Stock-II and stored at -20°C. The IRL-1620 stock solution was used for up to 30 days and was properly stored at -20°C.

[0073] Formulations and Dosage Forms

[0074] Formulations containing a base composition that promotes wound healing and nerve regeneration, with or without stem cells, are referred to as endothelin B receptor agonists.

[0075] In some embodiments, the base composition for promoting wound healing and nerve regeneration may be synthesized into various formulations and / or particles, as described below.

[0076] The composition may be formulated to take the form of a gel, cream, lotion, ointment, solution, solid, adhesive patch with a reservoir (allowing for sustained release of the composition), etc., and may be rubbed or sprayed onto the affected area, including the skin, subcutaneous tissue, muscle, fascia, blood vessels, and nerves, in need of regeneration or healing.

[0077] In some embodiments, the formulation may comprise a water-soluble gel polymer prepared from an effective amount of fibronectin. Examples of such compounds include vinyl polymers, e.g., polyacrylic acid, polyoxyethylene-polyoxypropylene block copolymers, e.g., poloxamer, and cellulose derivatives, e.g., hydroxypropyl cellulose (HPC). The polymer provides a viscosity of 50,000 to 1,000,000 cps at room temperature. The cream formulation is prepared from a commercially available cream base, i.e., Schering base (Schering Canada Inc., Point-Claire, Quebec), and has a viscosity of 60,000 to 80,000 cps at room temperature. The sustained-release system of the gel formulation provides sustained release of fibronectin to the wound site. This characteristic of these formulations reduces the frequency of wound application and reduces interference with the healing process. Such formulations maintain fibronectin delivery for up to 24 hours. Based on kinetic data, a twice-daily treatment schedule is the preferred embodiment.

[0078] In some embodiments, the delivery form may comprise an aqueous mineral oil emulsion that is easily spreadable, film-forming, and forms a stable, uniform layer upon application to moist skin or mucosal surfaces.

[0079] In some embodiments, the delivery form may comprise a drug-containing dissolvable sponge, such as gelfoam, which may be implanted subcutaneously, in a wound bed, or prior to closure of an incisional defect during surgery.

[0080] For specific nerve tissue regeneration, preferred embodiment delivery modes may include the use of a structure or "bridge" that traverses the gap length from the severed proximal nerve stump to either the distal nerve or the target tissue itself. In animal studies, various materials used to bridge the gap include peripheral nerve grafts, mesothelial cells, Millipore, and silicone tubing. Nerve guide tubes can be made of either silicone or biodegradable materials. Nerve guide tubes can be filled or coated with the compositions described in the preferred embodiments. Conduit designs can be fabricated into autograft-like structures that incorporate the compositions of the preferred embodiments. The use of biopolymers, cells, growth factors, and physical stimuli can be incorporated into the conduit to provide one or more guidance cues. For example, laminin-chitosan-PLGA nerve conduits can be supplemented with the compositions of the preferred embodiments and stem cells to promote recovery of injured nerves.

[0081] In some embodiments, the formulation comprises a biodegradable polymer. In further embodiments, the biodegradable polymer is poly(lactic-co-glycolic acid) (PLGA) or PEGylated PLGA (PEG-PLGA). In some embodiments, the composition may comprise additional additives, such as, but not limited to, polyvinyl alcohol (PVA) or other known nanoparticle stabilizers. In some embodiments, the nanoparticles are magnetically responsive or comprise magnetically responsive particles. In some embodiments, the magnetically responsive particles are optionally superparamagnetic iron oxide (SPION). Finally, in some embodiments, the nanoparticles may be further comprised in a solution, suspension, gel, or other formulation suitable for their delivery.

[0082] Embodiments can be formulated to facilitate release timing. For example, a poorly water-soluble second drug (e.g., TIDE) can be encapsulated within the organic shell of ETBRA, with or without stem cells loaded with a hydrophilic drug that regenerates damaged tissue.

[0083] Our preferred embodiments for topical drug delivery are niosome gels and thin film hydrogels.

[0084] Thin Film Hydrogel Embodiments

[0085] The hydrogel developed for the experimental purpose was a thin film of calcium alginate hydrogel. The development procedure is as follows.

[0086] (material and method)

[0087] (Estimation of IRL-1620 in hydrogel samples by HPLC analysis)

[0088] Approximately 10 mg of hydrogel sample was weighed into a centrifuge tube. 10 ml of phosphate buffer (pH 7.4) was added to the tube and sonicated at 2-8°C for 30 minutes. The tube was then centrifuged at 5,000 rpm and 5°C, and the supernatant was collected. Next, 0.5 ml of the supernatant was transferred to a 10 ml volumetric flask, and the volume was adjusted with a diluent (acetonitrile:water = 40:60, v / v). The sample was used for HPLC analysis.

[0089] (Preparation of Alginate-Based Films)

[0090] Alginate films were prepared using a solvent casting process (Pereira et al., 2013). A solution of sodium alginate (1.5%, w / v) was prepared by dissolving the powder in distilled water under mechanical stirring at 600 rpm until a homogeneous solution was formed (approximately 2 h). During the preparation of the alginate solution, the plasticizer glycerol was added at a ratio of 15% (w / w) of the alginate powder weight. IRL-1620 was dispersed in 1 ml of water and stirred at 600 rpm in an ice bath. The solution was then transferred to an ultrasonic system (30 min) and placed under controlled temperature (2–8 °C). It was then degassed in a refrigerator at 2–8 °C for 12 h. Pure alginate films with glycerol were prepared by casting 5 mL of the solution onto a Petri dish (o = 5 cm). The film was allowed to stand for 5 min. Next, 2 mL of 5.0% (w / v) CaCl2 aqueous solution was poured into the Petri dish and gently stirred to ensure uniform dispersion of the CaCl2 and allow cross-linking for 15 min. The hydrogels were then washed with distilled water, sterilized in a clean bench under UV irradiation, and stored frozen for 48 h. The films were then lyophilized to constant weight using a freeze dryer (Allied Frost) (DiMicco et al., 2007).

[0091] (Compatibility Test)

[0092] The compatibility of IRL-1620 with the excipient sodium alginate was evaluated using the FTIR peak matching method (Riaz & Ashraf, 2015) using a Bruker FTIR (Alpha-1) in the range of 4,000 cm-1 to 650 cm-1.

[0093] (SEM test)

[0094] The surface morphology of samples containing different concentrations of sodium alginate was investigated by scanning electron microscopy (SEM LEO43 SVP, Cambridge) ( Hashem et al., 2013 ).

[0095] (film thickness)

[0096] The film thickness was measured at five different locations on the film with a caliper micrometer to an accuracy of 0.001 mm, and the results were expressed as the mean ± standard deviation (SD) of the measurements.

[0097] (film light transmission and transparency)

[0098] The light transmittance and transparency of the films were measured with a spectrophotometer according to the method of Norajit et al. Rectangular samples (10 mm × 35 mm) of the films were cut and placed in a spectrophotometer cell to measure the light blocking properties in the wavelength range comprised of 200–800 nm, with air used as the control. The transparency of the films was calculated using the following equation:

[0099] Transparency=Abs 600 / x

[0100] where Abs 600 represents the absorbance value at 600 nm, and x corresponds to the film thickness in millimeters (Norajit & Ryu, 2012).

[0101] (water soluble)

[0102] It is widely recognized that water solubility and swelling are important properties in the characterization of biodegradable materials used in tissue regeneration and drug delivery applications.

[0103] To measure water solubility, samples of cross-linked hydrogel alginate films were freeze-dried and immersed in 40 mL of distilled water with stirring at 120 rpm for 24 hours. The samples were then removed from the medium, excess water removed with filter paper, and the wet weight was immediately calculated to determine water absorption. The samples were then dried in an oven at 37 °C to constant weight, and the dissolved mass was determined.

[0104] The water absorption rate and water solubility rate were calculated using the following formulas.

[0105] Water absorption rate (%)=[(Ww-Wd) / Wd]×100

[0106] Water dissolution rate (%)=[(Wi-Wf) / Wi]×100

[0107] where Ww represents the wet weight of the film, Wd corresponds to the dry weight of the film, Wi represents the initial dry weight of the film before immersion in distilled water, and Wf corresponds to the dry weight of the film after immersion (Brown et al., 2011).

[0108] (Swelling behavior)

[0109] To determine the swelling behavior of the hydrogel films, small rectangular pieces of film measuring 1 x 1 cm were cut and weighed. The films were placed in a Petri dish containing 20 ml of phosphate-buffered saline (pH 7.4). The hydrogels were removed from the buffer after 30 min and reweighed. Therefore, the swelling behavior was measured as the "% swelling index."

[0110] % Swelling Index (%) = [(Wh-Wd) x 100] / Wd

[0111] Wh represents the weight of the product after hydration for 30 minutes, and Wd represents the weight of the dry product (SW Kim et al., 1992).

[0112] (Biodegradability test)

[0113] A small piece of film (2 x 2 cm) was placed in 25 ml of phosphate buffer at room temperature for 7 days, and the film was visually observed for dissolution.

[0114] (In vitro drug release test)

[0115] In vitro dissolution testing of the hydrogel films was performed using goat skin. Fresh goat skin was obtained from a local slaughterhouse. It was washed and shaved. IRL-1620 permeability was assessed using Franz diffusion cells at 100 rpm and 37 ± 5°C using 50 ml of phosphate-buffered saline, pH 7.4, as the dissolution medium. 0.5 ml aliquots were withdrawn at predetermined time intervals up to 48 h, and the IRL-1620 concentration was analyzed by HPLC. After each sampling, an equal volume of fresh medium was replenished to maintain a constant volume (Thu et al., 2012). Samples were diluted with 0.5 ml of acetonitrile:water (40:60, v / v) and analyzed by HPLC.

[0116] (Stability test)

[0117] The stability of the hydrogel film formulation was evaluated by keeping it in a sealed glass vial and storing it under three storage conditions for 45 days: frozen (-20°C), refrigerated (2-8°C), and room temperature (Sabale & Vora, 2012). Samples were taken at different time intervals during the study period, and % film quantification was performed using HPLC.

[0118] (result)

[0119] (Drug Analysis)

[0120] The high-performance liquid chromatography (HPLC) method developed for the quantification of IRL-1620 was simple and sensitive. Separation was performed on a reversed-phase C18 column (250 mm × 4.6 mm), and the column effluent was monitored with a UV detector at 215 nm. This study demonstrated that the method is precise, accurate, and specific. This method was successfully applied to encapsulation efficiency, drug release, and stability studies (Figure 2).

[0121] (Preparation of Hydrogel Films)

[0122] Addition of calcium chloride solution resulted in the formation of calcium alginate, yielding a transparent film (Figure 2). The film was allowed to harden for 15 minutes, yielding a hardened, uniform, continuous, and flexible hydrogel film (Figure 3). These films were stored frozen and then freeze-dried. After freeze-drying, a thin, white hydrogel film was obtained (Figure 5). The surface was smooth, flexible, and not brittle.

[0123] (Compatibility Test)

[0124] No appearance or disappearance of peaks in the IR spectrum was observed in the drug-polymer mixture, confirming the absence of any chemical interaction between the drug and sodium alginate, as shown in Figures 6 to 8.

[0125] (Surface morphology observation by SEM)

[0126] The surface morphology of the freeze-dried hydrogel films was evaluated by SEM observation. This was also performed for sodium alginate films with different concentrations (Figure 9). The best morphological observation was shown in Figure 9(d), and thus this was used for further study. The 1.5% sodium alginate film had a smooth and uniform texture, indicating good cross-linking of the alginate groups. At concentrations of 1% and 1.25%, brittle films with numerous cracks were obtained. At higher concentrations (2%), sodium alginate was observed to form rough films.

[0127] (film thickness)

[0128] The fabricated hydrogel films exhibited smooth surfaces, high plasticity, and thicknesses ranging from 110 to 120 μm (Table 2). Because film thickness affects in vivo dissolution in the wound area, a low concentration of sodium alginate was selected (Tokarev & Minko, 2010). This was sufficient to maintain the film's mechanical strength and reduce its brittleness.

[0129] [Table 2]

[0130] (film light transmission and transparency)

[0131] The properties of light transmission and transparency are important parameters for evaluating the quality and effectiveness of films in topical wound healing (Norajit & Ryu, 2012). Low concentrations of IRL-1620 did not affect the transparency of the films.

[0132] (water soluble)

[0133] The aqueous solubility and swelling behavior of the hydrogel were investigated to evaluate the behavior of the film after 24 hours. The aqueous solubility was approximately 8.32% (Table 2). Therefore, the film can be placed in an open wound for an extended period of time.

[0134] (Swelling behavior)

[0135] The swelling behavior of the hydrogel film was found to be 146.8%. This high swelling capacity can be attributed to the hydrophilic functional groups, such as NH2- and COO-, derived from the alginate polymer

[63] . Therefore, the developed hydrogel film can be used as a wound healing agent with a high aqueous solution absorption capacity. Furthermore, it also contributes to the absorption of wound exudate.

[0136] (Biodegradability test)

[0137] The samples were observed daily for dissolution. The films immediately swelled and gradually decreased in size each day. Finally, a homogeneous solution was formed on the seventh day of testing. Thus, it took seven days for the films to decompose and release alginate into the phosphate buffer solution.

[0138] (In vitro drug release test)

[0139] In vitro testing of the alginate hydrogel films was performed through goat skin using Franz diffusion cells in PBS pH 7.4, and the results are shown in Figures 11 and 12. The data show that the in vitro drug release of IRL-1620 from the hydrogel film formulation increased rapidly up to 5 hours, then maintained at a constant level for approximately 24 hours, with a rapid decrease in release observed after 26 hours. The cumulative release of IRL-1620 from the formulation was 86.54% after 24 hours (Figure 13).

[0140] (Stability test)

[0141] Stability studies were performed on the alginate film formulations by storing them under different storage conditions for 45 days. Samples stored at room temperature showed changes in appearance. The color of the films changed after 15 days of storage, and they may be considered unstable at room temperature. Refrigerated and frozen samples showed physical stability, and thus, quantification of IRL-1620 was performed by HPLC, and the results are shown in Figure 14.

[0142] The results showed that the drug content decreased at 2 to 8°C, but no significant change was observed when frozen (-20°C). Therefore, it was found that the developed hydrogel formulation was stable at the end of the test when frozen (-20°C).

[0143] (Conclusion)

[0144] An alginate-based hydrogel dressing was successfully developed by solvent casting using glycerol as a plasticizer and calcium chloride as a crosslinker. A 1.5% sodium alginate concentration provided a hydrogel film with a smooth surface and good mechanical strength. The hydrogel's swelling factor and water solubility indicate its suitability for long-term wound healing. IRL-1620 release was observed for up to 24 hours; however, the alginate film remained in contact with the tissue, allowing for continued wound healing by retaining moisture at the affected site. The formulation was found to be stable at -20°C.

[0145] Niosome Gel Embodiments

[0146] Another alternative to this preferred embodiment are niosome gels that have both hydrophilic and hydrophobic properties, which, due to the presence of cholesterol, prolong the release of the encapsulated drug and allow penetration into the epithelial layer.

[0147] (Estimation of IRL-1620 in niosome samples by HPLC analysis)

[0148] Approximately 1.0 mg of niosomes was accurately weighed into a 10 ml volumetric flask. 0.5 ml of butanol was added and the mixture was shaken to completely wet the niosomes. This disrupted the cholesterol and soy lecithin membranes. The dilution solvent was added to adjust the volume to 10 ml. The sample was analyzed by HPLC.

[0149] (Estimation of IRL-1620 in niosome gel samples by HPLC)

[0150] 100 mg of niosome gel was accurately weighed and dispersed in 10 ml of phosphate buffer (pH 6.8) and centrifuged at approximately 3000 rpm. The supernatant was removed, and the precipitated residue was treated with 0.5 ml of butanol and adjusted to a volume of 10 ml with acetonitrile:water (40:60, v / v). The sample was analyzed by HPLC.

[0151] (Preparation of niosomes)

[0152] IRL-1620 was dissolved in ethanol and used to prepare niosomes. Niosome complexes (1:1, 1:2, 1:3, 1:4, and 1:5 drug-to-soy lecithin ratios) were prepared using ethanol as the reaction medium, with the cholesterol concentration kept constant. Soy lecithin and cholesterol were dissolved in 10 ml of dichloromethane (Pando et al., 2013) and added dropwise to 5 ml of ethanol solution containing IRL-1620 (20 μg / ml) with continuous stirring and sonication for 15 min. The resulting mixture was evaporated under reduced pressure at 2–8 °C. The residue (niosomes) was placed in a desiccator. The niosomes were stored at 2–8 °C.

[0153] [Table 3]

[0154] (Characterization of niosome complexes)

[0155] The complexes were characterized using an optical microscope. The complexes were suspended in buffer, a drop was placed on a slide, and covered with a coverslip. The complexes were viewed under a microscope at 10 × 10 magnification (Junyaprasert et al., 2008).

[0156] (Encapsulation efficiency (EE))

[0157] Ten mg of niosomes were added to 10 ml of phosphate buffer (pH 7.4) and centrifuged at 12,000 rpm for 45 minutes using a Remi centrifuge to separate the niosomes from unencapsulated drug. The separated precipitated niosomes were treated with 0.5 ml of butanol and the volume was adjusted to 10 ml with acetonitrile:water (40:60, v / v). The IRL-1620 concentration in the supernatant was measured by HPLC. The drug encapsulation rate was calculated using the following formula (Singh et al., 2011):

[0158] Encapsulation efficiency (%) = (total drug weight - free drug weight) × 100 / total drug weight

[0159] (Compatibility Test)

[0160] The compatibility of IRL-1620 with the excipients soy lecithin and cholesterol was evaluated using the FTIR peak matching method (Lo'pez-Lorente & Mizaikoff, 2016). The instrument used was a Bruker FTIR (Alpha-1) with a measurement range of 4,000 cm. -1 ~650cm -1 It was.

[0161] (particle size analysis)

[0162] To keep the niosomes dispersed during size measurement, a magnetic stirring cell dispersion unit (Malvern Instruments Ltd., UK) was used with medium stirring speed. The measurement position was 1.05 mm, and the polydisperse mode of analysis was selected. These conditions allowed for accurate measurement of particles in the range of 0.1 nm to 10,000 nm

[65] .

[0163] (Zeta potential analysis)

[0164] The importance of zeta potential is that its value can be related to the stability of a colloidal dispersion. The zeta potential value indicates the degree of repulsion between like-charged niosomes in the dispersion. The zeta potential of niosome dispersions was also measured with a Malvern instrument

[66] .

[0165] (Development of topical gel for niosomes)

[0166] Topical gels for niosomes were formulated using hydroxyethylcellulose (HEC), triethanolamine (TEA), polyethylene glycol 400 (PEG), methylparaben, and water. Different concentrations of HEC were dispersed in water to determine the optimum concentration for gel formation. The ideal ratio was approximately 11% (w / v) solution. TEA was used as a surfactant and PEG as a stabilizer to disperse the hydrophobic niosomes in the HEC gel base. Various combinations are shown in Table 4.

[0167] [Table 4]

[0168] (Details of excipients)

[0169] Hydroxyethyl cellulose is a partially substituted poly(hydroxyethyl) ether of cellulose, a non-ionic hydrophilic polymer with a linear filament structure and a minimum degree of substitution of 1.5 (three hydroxyl groups substituted every two units)

[67] .

[0170] Triethanolamine is ammonia with each hydrogen replaced by a 2-hydroxyethyl group. It acts as a buffer and surfactant. It is a tertiary amino compound, a triol, and an amino alcohol. It is derived from triethylamine. It is the conjugate base of triethanolammonium.

[0171] PEG 400 is a low molecular weight grade polyethylene glycol. It is a clear, colorless, viscous liquid. Due to its low toxicity, PEG 400 is widely used in various pharmaceutical formulations

[68] .

[0172] Methylparaben is a 4-hydroxybenzoic acid ester derived from the condensation of the carboxyl group of 4-hydroxybenzoic acid with methanol and formaldehyde. It is the most frequently used antimicrobial preservative in cosmetics. In addition, it also acts as a plant metabolite, antimicrobial food preservative, neuroprotectant, and antifungal agent

[69] .

[0173] (Evaluation of niosome gel)

[0174] The gel formulations were evaluated based on the following parameters:

[0175] Clarity: Determined by visual inspection under black and white background and graded as hazy, clear, very clear (glassy).

[0176] Uniformity: The appearance of the gel was determined by visual inspection.

[0177] Sedimentation: The presence or absence of aggregates was determined by visual inspection.

[0178] In vitro drug release test

[0179] All prepared gels were subjected to in vitro dissolution testing in goat skin Franz diffusion cells at 100 rpm and 37 ± 5°C using 50 ml of phosphate-buffered saline, pH 7.4, as the dissolution medium (Fathalla, 2014) (Helal et al., 2012). 0.5 ml aliquots were withdrawn at predetermined time intervals up to 48 h and analyzed for IRL-1620 concentration by HPLC. After each sampling, the volume was kept constant with an equal volume of fresh medium. The samples were then mixed with 0.5 ml of acetonitrile:water (40:60, v / v) and subjected to HPLC analysis.

[0180] (Stability test)

[0181] The stability of the niosome gel formulation was evaluated by keeping it in a sealed glass vial and storing it under two storage conditions, namely, refrigerated temperature (2-8°C) and room temperature for 45 days (Dantas et al., 2016). Samples were taken at different time intervals during the study period and % gel quantification was performed by HPLC.

[0182] (result)

[0183] (Analysis of IRL-1620 (Sobateltide))

[0184] The high-performance liquid chromatography (HPLC) method developed for the quantification of IRL-1620 was simple and sensitive. Separation was performed on a reversed-phase C18 column (250 mm × 4.6 mm), and the column effluent was detected by UV at 215 nm. The method exhibited excellent precision, accuracy, and specificity throughout the study period. This method was successfully applied to encapsulation efficiency, drug release, and stability studies.

[0185] (Characterization of niosome complexes)

[0186] Microscopic images of the niosome complexes observed at 10 × 10 magnification showed the presence of oval to spherical vesicles.

[0187] (Encapsulation efficiency (EE))

[0188] Drug encapsulation studies showed that maximum drug encapsulation was observed in F3. The encapsulation efficiency of all formulations is shown graphically in Figure 17.

[0189] The prepared niosomes showed high encapsulation efficiency. The F3 formulation had the highest encapsulation efficiency of IRL-1620 at 93%, indicating the optimal lipid amount required for niosome formation. Further increase in lipid concentration did not significantly change the encapsulation efficiency, indicating that an increase in lipid concentration does not contribute to the encapsulation of the drug within the matrix.

[0190] [Table 5]

[0191] (Compatibility Test)

[0192] No appearance or disappearance of peaks in the IR spectra was observed in the drug-lipid mixtures, confirming the absence of any chemical interaction between the drug and lipids, as shown in Figures 18-20.

[0193] (particle size analysis)

[0194] Particle size analysis showed that the prepared niosomes had particle sizes in the range of 218 nm (Figure 21) and increasing the ratio of soybean lecithin did not change the size of the niosomes.

[0195] (Zeta potential analysis)

[0196] The zeta potential value of IRL-1620-loaded niosomes was -35.1 mV, which is an indicator of the net charge of the niosomes (Figure 22). This negative charge on the surface of niosomes creates repulsion between particles, enhancing stability and preventing aggregation (Bayindir & Yuksel, 2010). Therefore, the niosomes have acceptable stability.

[0197] (Evaluation of niosome gel)

[0198] The results of appearance, color, uniformity and sedimentation were observed as shown in Table 6. G-3 was considered to have the best combination in terms of texture, uniformity and spreadability.

[0199] [Table 6]

[0200] (In vitro drug release test)

[0201] In vitro testing of the niosome gel formulation (Shahiwala & Misra, 2002) was performed through goat skin using Franz diffusion cells in PBS pH 7.4, and the results are shown in Figure 24. The data showed that the in vitro drug release of IRL-1620 from the niosome gel formulation increased sharply up to 6 hours, after which the concentration was maintained for approximately 26 hours, and the release gradually decreased. The cumulative release of IRL-1620 from the formulation was 81.1% after 24 hours (Figure 25).

[0202] (Stability test)

[0203] Stability studies were carried out on the niosome gel formulations by storing them under different storage conditions for one month. Samples were taken at different time points and quantified for IRL-1620 by HPLC method, and the results are shown in Figure 26.

[0204] The results showed that there was a decrease in drug content at room temperature, whereas no significant change was observed at refrigerated temperature.Therefore, the developed niosome formulation was found to be stable under storage conditions at the end of the study.

[0205] (Conclusion)

[0206] The encapsulation efficiency of IRL-1620 niosomes ranged from 6.99% to 93.0%. The niosome formulations exhibited particle sizes in the 218 nm range. Compatibility, zeta potential, and release test data indicated that encapsulating drugs in niosomes can sustain and control drug release over a long period of time and improve stability. The use of hydroxyethyl cellulose and triethanolamine as gel bases improved the dispersibility and release of IRL-1620 from niosomes. Therefore, the developed formulation has the ability to encapsulate IRL-1620, achieve long-term drug release, and enhance the stability of the peptide moiety.

[0207] (test)

[0208] In any of the above embodiments, tests are required to quantify the regeneration of injured tissue. Granulation and epithelialization of the wound bed can be strategically quantified to assess wound healing. Microscopy, such as electron microscopy, can be used to measure in vitro tissue samples to quantify neuroepithelial and epithelial structures. The two-point discrimination test can be used to measure sensory loss. Thermal stimulation of the skin can also be used to test nerve recovery. Other tests that directly measure nerve conduction, such as electromyography and nerve conduction velocity testing, can also be used.

[0209] A comparative study of two preferred embodiments was conducted on acute wound healing.

[0210] Title: Pharmacological evaluation of the wound-healing activity of topical gels and biodegradable hydrogel films in excision and incision wound models in rats

[0211] Study Details: This study was conducted to examine the effects of treatment on wound healing in rats. The study included excision and incision models of wound healing.

[0212] List of abbreviations: IHC: immunohistochemistry; MT: Masson's trichrome; TEM: transmission electron microscopy; bFGF: basic fibroblast growth factor; PDGF: platelet-derived growth factor.

[0213] GLP Compliance Statement: This study was conducted in accordance with current principles of Good Laboratory Practice (GLP).

[0214] (Excision model)

[0215] the purpose: Excisional wounds will be induced and the wound healing activity of the test product will be evaluated in comparison with the reference product based on the following: ·Wound area ·Epithelialization time Wound contraction rate ·Wet granulation tissue weight H&E, MT staining and IHC

[0216] (material and method)

[0217] Animals: The experimental protocol for this study followed the guidelines of the Committee for the Control and Supervision of Animal Experiments (CPCSEA, Delhi, India) and was reviewed and approved by the Institutional Animal Ethics Committee of DPSRU, New Delhi. Albino Wistar rats weighing 180–200 g were used. The animals were acclimatized in the institutional animal care room for one week before use. The animals were allowed free access to food and water.

[0218] [Table 7]

[0219] Animals were allowed to acclimate for 7 days before the start of the study. Wistar albino rats were anesthetized with ketamine hydrochloride (50 mg / kg, intraperitoneal administration), and the dorsal skin was shaved with depilatory cream and wiped with 70% ethanol. A standard ring was placed approximately 500 mm behind the rat. 2 The area was marked, and the marked skin was carefully excised in full thickness with a scalpel. In the placebo 1 and treatment 1 groups, rats received topical application of placebo 1 and gel, respectively, for 16 days. Placebo 2 and biodegradable hydrogel film were applied every 3 days, and the films were covered with cotton and gauze in the placebo 2 and treatment 2 groups, respectively. The positive control group received standard medication. In the treatment 1 + STD and treatment 2 + STD groups, standard medication and each test medication were applied in combination. The day of wound induction was designated as day 0. The wound area was measured on days 4, 8, 12, and 16, and the wound contraction rate was calculated. On study day 17, animals were euthanized and tissues were collected for H&E, IHC, and MT staining.

[0220] observation:

[0221] On day 5, no epithelialization was observed in the skin.

[0222] Wound size was observed on days 4, 8, 12, and 16 (Table 7).

[0223] The day when the scab fell off without leaving an unhealed wound was observed (Table 8).

[0224] The following parameters were observed:

[0225] [Table 8]

[0226] All data are expressed as mean ± SD. *p≦0.05, **p≦0.01, ***p≦0.001 compared with disease controls; #p≦0.05, ###p≦0.001 compared with placebo 1; @p≦0.05, @@@p≦0.001 compared with placebo 2.

[0227] Statistical analysis report: Overall, treatment groups showed improved wound contraction rates compared to their respective placebo groups. The positive control significantly improved wound contraction rates compared to disease controls. However, treatment 2 (film) did not show any improvement in wound contraction rates compared to placebo 2 (film), but showed a significant effect on wound contraction rates when used in combination with standard medication.

[0228] (2) Epithelialization period

[0229] [Table 9]

[0230] All data are expressed as mean ± SD. *p < 0.05 compared with disease control; #p < 0.05 compared with placebo 1; @p < 0.05 compared with placebo 2.

[0231] Statistical analysis report: Standard medication significantly improved the epithelialization rate compared to disease controls. Each treatment significantly improved the epithelialization rate compared to its respective placebo. However, treatment 2 (film) did not improve the epithelialization period compared to placebo 2 (film), but when used in combination with standard medication, it showed a significant effect on the epithelialization period.

[0232] (3) Wet granulation tissue weight

[0233] On the advice of a veterinarian, wet granulation tissue weight was not evaluated in this study because collection of granulation tissue may affect H&E results.

[0234] In Figure 27, "II" indicates inflammatory cell infiltration, "B" indicates blood vessels, "An" indicates angiogenesis, "FP+" indicates fibroblast proliferation, "E" indicates epidermis, and "H" indicates histiocytes. "A, B, C, D, E, F, G, and H" indicate disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0235] (Histological evaluation)

[0236] Histological evaluation of the skin was performed by H&E staining. Neutrophil infiltration and dense inflammatory cell infiltration (II) were observed in the placebo 1, placebo 2, and disease control groups. However, moderate inflammation was observed in the treatment groups. Neovascularization, fibroblast proliferation, and collagen re-epithelialization were not observed in the placebo 1, placebo 2, and disease control groups. Various treatments improved angiogenesis, fibroblast proliferation, and collagen deposition (Figure 27).

[0237] In Figure 28, "C" represents collagen, "FC" represents fibrous connective tissue, and "A, B, C, D, E, F, G, H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0238] MT staining: A mild increase in collagen deposition was observed in the disease control, placebo 1, and placebo 2 groups, while improvements were observed in each treatment group. Increased collagen deposition was observed throughout the dermis in the positive control, treatment 1 + STD, and treatment 2 + STD groups (Figure 28).

[0239] In Figure 29, "A, B, C, D, E, F, G, H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0240] In Figure 30, "A, B, C, D, E, F, G, H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0241] Immunohistochemistry: In placebo 1, negative expression of VEGF was observed in the fibrotic areas throughout the dermis. However, weak VEGF positivity was observed in the cytoplasm of vascular endothelial cells and some spindle cells in placebo 2 and disease control. Higher levels of VEGF expression were observed in the treatment groups. Furthermore, weak vWF expression in vascular endothelial cells throughout the fibrotic areas of the dermis was observed in placebo 1, placebo 2, and disease control groups (Figure 29). All treatments improved vWF expression, but strong vWF positivity in the cytoplasm of vascular endothelial cells was observed in treatment 2, treatment 1 + STD, and treatment 2 + STD (Figure 30).

[0242] Conclusions: The various treatments improved the wound healing process by improving inflammatory parameters, fibroblast proliferation, and angiogenesis. The treatments improved the rate of wound contraction and epithelialization. The treatments accelerated the wound healing process by increasing collagen deposition and enhancing VEGF and vWF expression compared with placebo 1, placebo 2, and disease control groups.

[0243] (Incision model)

[0244] the purpose: Inducing an incision wound and applying the test drug -Measure wound breaking strength (WBS) - Collecting tissues from animals for H&E, IHC, and MT staining

[0245] (material and method)

[0246] Animals: The experimental protocol for this study followed the guidelines of the Committee for the Control and Supervision of Animal Experiments (CPCSEA, Delhi, India) and was reviewed and approved by the Institutional Animal Ethics Committee of DPSRU, New Delhi. Albino Wistar rats weighing 180–200 g were used. The animals were acclimatized in the institutional animal care room for 1 week before use and allowed free access to food and water.

[0247] [Table 10]

[0248] method: Animals were allowed to acclimate for 7 days before the start of the study. Wistar albino rats were anesthetized with ketamine hydrochloride (50 mg / kg, intraperitoneal injection), and the dorsal skin was shaved with electric clippers (10 watts) and cleaned with 70% ethanol. Two 6-cm parallel paraspinal incisions were made 1 cm lateral to the spinal midline, penetrating the full thickness of the skin. In the placebo 2, treatment 2, and treatment 2 + STD groups, placebo film, biodegradable film, and biodegradable film + Regen D were applied, respectively. The wound was closed with surgical sutures at 1cm intervals. The sutures were removed on the 7th day after wound placement. The placebo 1, positive control, treatment 1, and treatment 1 + STD groups received placebo gel, standard drug Regen-D, test gel, and test gel + Regen-D, respectively, for 16 days. The day of wound induction was designated as day 0. On study day 17, animals were euthanized and tissues were collected for H&E, IHC, and MT staining.

[0249] observation: On day 7, copious pus formation was observed in animals 1, 2, and 3, and in animals 1 and 2 of placebo 1 and treatment 2, respectively. On day 7, copious pus formation was observed in animals 1, 2, and 3 of Treatment 2 + STD. On day 10, copious pus formation was observed in animal 3 of treatment 2. Wound breaking strength was measured on the 10th day of the test (Table 9).

[0250] The following parameters were observed:

[0251] [Table 11]

[0252] All data are expressed as mean ± SD. ***p≈0.001 compared with disease controls; #p≈0.05 compared with placebo 1; @p≈0.05, @@@p≈0.05 compared with placebo 2.

[0253] Statistical analysis report: Both treatments improved wound breaking strength compared to their respective placebos. However, the standard group improved wound breaking strength compared to disease controls.

[0254] Histological evaluation (H&E)

[0255] In Figure 32, "II" indicates inflammatory cell infiltration, "B" indicates blood vessels, "An" indicates angiogenesis, "FP+" indicates fibroblast proliferation, "E" indicates epidermis, and "H" indicates histiocytes. "A, B, C, D, E, F, G, and H" indicate disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0256] Histological evaluation: Histological evaluation was performed by H&E staining. Increased inflammatory parameters were observed in the placebo 1, placebo 2, and disease control groups, while various treatments reduced inflammation compared to the placebo 1 and placebo 2 groups. However, mild inflammation was observed in the treatment 1 group. Treatment increased angiogenesis and fibroblast proliferation in each group. Furthermore, normal epidermis was observed in the treatment 1 + STD and treatment 2 + STD groups (Figure 32).

[0257] In Figure 33, "C" represents collagen, "FC" represents fibrous connective tissue, and "A, B, C, D, E, F, G, H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0258] MT staining: A slight increase in collagen deposition was observed in the placebo 1, 2, and disease control groups. Collagen deposition was slightly higher in the other groups compared to the placebo 1, placebo 2, and disease control groups (Figure 33).

[0259] In Figure 34: "A, B, C, D, E, F, G, H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, treatment 2 + STD, respectively.

[0260] In Figure 35: "Immunohistochemistry: Assessment of vWF expression"

[0261] and "A, B, C, D, E, F, G, H" indicate disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1 + STD, and treatment 2 + STD, respectively.

[0262] Immunohistochemistry: Weak cytoplasmic positivity for VEGF expression was observed in vascular endothelial cells in the placebo 1, placebo 2, positive control, treatment 1, and treatment 2 + STD groups, but no VEGF expression was observed in vascular endothelial cells in the placebo 2 and disease control groups. Increased VEGF expression was observed in the treatment 2 and treatment 1 + STD groups (Figure 8). No vWF expression was observed in the placebo 2 and disease control groups. Weak cytoplasmic positivity for vWF was observed in vascular endothelial cells in the placebo 1, positive control, treatment 1, treatment 2, and treatment 1 + STD groups, but vWF expression was higher in the treatment 1 + STD group compared to the other groups (Figure 35).

[0263] Conclusions: Treatment improved fibroblast proliferation, collagen deposition, and angiogenesis. Treatment slightly improved VEGF and vWF levels in each group. Treatment significantly improved various parameters compared with the placebo group. Furthermore, the standard group significantly increased WBS compared with normal controls.

Claims

1. A method for treating skin or cutaneous wounds and injured cranial or peripheral nerves with compounds containing endothelin analogs that limit cell death through mechanisms of anti-apoptotic, anti-inflammatory, and antioxidant activity, and promote the formation of new nerve cells (neurogenesis) and new blood vessels (angiogenesis) to enhance wound healing and regenerate nerves.

2. The endothelin B receptor analog is N-succinyl-[Glu ９ , Ala １１，１５ ] Endothelin 1 (sovateltide, IRL-1620), BQ-3020, [Ala １，３，１１，１５ ]-endothelin, sarafotoxin S6c, and endothelin 3.

3. (a) Endothelin-B (ET Ｂ ) receptor agonists, (b) one or more components selected from the following: growth factors, osmotic agents such as glycerol, diuretics such as furosemide or ethacrynic acid, steroids, transplanted stem cells, ferulic acid, arctigenin, espin isoform 1 polypeptide, neurotrophic factors selected from the group consisting of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, and ciliary neurotrophic factor, human insulin-like growth factor 1, netrin-1, and mixtures thereof; and (c) optionally an excipient.

4. (a) Endothelin-B (ET Ｂ a packaged composition comprising a receptor agonist and a steroid or a salt thereof; (b) Said ET Ｂ a document containing instructions for administering the receptor agonist and the steroid or salt thereof simultaneously or sequentially to treat a patient; (c) a container for (a) and (b); 1. An article of manufacture comprising:

5. The endothelin-B receptor agonist is N-succinyl-[Glu ９ , Ala １１，１５ ] The article of claim 4, wherein the compound is endothelin 1 (sovateltide, IRL-1620).

6. 3. The method of claim 2, wherein the endothelin B receptor analog is delivered topically, subdermally, subcutaneously, intramuscularly, intravenously, or orally.

7. The method of claim 2, wherein the endothelin B receptor analog by itself or nanoparticles, including stealth nanoparticles, combined with a hydrogel are prepared into various formulations such as gels, creams, solutions, lotions, ointments, adhesive patches, and sprays.

8. The method of claim 2, wherein the endothelin B receptor analog is administered via a sustained-release drug delivery system such as Gelfoam®, hyaluronic acid gel, Seprapack™, thiol-modified hyaluronic acid, glutaraldehyde-crosslinked porcine collagen, gelatin, chitosan sugar chain derivatives, Poloxamer 407, or chitosan glycerophosphate hydrogel, and the sustained-release system is lipid-core nanocapsule poly-L-lysine (HBPL) nanoparticles.

9. 2. The method of claim 1, wherein the endothelin analogue, particularly the endothelin B receptor analogue, is used for treating wounds and / or promoting wound healing and for regenerating damaged nerves in mammals.

10. The method of claim 1, wherein the administration of the endothelin analog may be combined with a growth factor, an osmotic agent such as glycerol, a diuretic such as furosemide or ethacrynic acid, a steroid, transplanted stem cells, ferulic acid, arctigenin, espin isoform 1 polypeptide, a neurotrophic factor selected from the group consisting of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5 and ciliary neurotrophic factor, human insulin-like growth factor 1, or netrin-1, or a mixture thereof.

11. 10. The method of claim 1, wherein the dosage range of the endothelin analog is 0.00001 to about 1 mg, and can be administered one or more times per day, several days, weeks, or months.

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