Compositions and methods for wound healing and damaged nerve repair

Abstract

Aspects of the claimed embodiments relate to compositions and methods for treating wounds and / or promoting wound healing and mammalian nerve regeneration of damaged nerves. Furthermore, the application relates to the use of a therapeutic amount of an endothelin-B receptor agonist. In particular, the present application relates to a method of treating dermal or skin wounds and damaged cranial or peripheral nerves using IRL-1620 (Sovastatin). In some embodiments, a composition may comprise an endothelin-B receptor agonist, which may comprise a peptide, such as Sovastatin (IRL-1620). Delivery systems comprising agents for topical applications, including gels, sponges, gauzes, and webs, are also described.

Description

TECHNICAL FIELD

[0001] The present application relates to methods and compositions for enhancing the rate and quality of wound healing, including promoting neurovascular and muscle tissue regeneration in an individual in need thereof. The methods include administering an endothelin B (ETB) receptor agonist and one or more bioactive factors or stem cells. BACKGROUND

[0002] Wound healing is a complex process involving multiple stages. These stages include: 1) coagulation that begins immediately after injury; 2) inflammation that begins a few minutes later; 3) migration and proliferation processes (granulation stage) that begin within hours to days; and 4) remodeling process, followed by the formation of full strength skin [1-3]. Coagulation controls hemostasis and initiates healing by releasing various growth factors and cytokines from degranulating platelets. During the inflammation stage, platelets aggregate and clot to form a matrix that traps plasma proteins and blood cells to induce the influx of various types of cells. Neutrophils are the first to arrive and function to phagocytose contaminating bacteria, digest fibrin clots, release mediators to attract macrophages, and activate fibroblast and keratinocyte functions [3]. Macrophages digest pathogens, debride the wound, and secrete cytokines / growth factors (e.g., interleukin-1 (I-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-beta), and basic fibroblast growth factor (bFGF)); these cytokines / growth factors stimulate fibroblasts and endothelial cells. Overall, the inflammation stage is important for preventing infection and promoting the migration and proliferation stages of wound healing.

[0003] There are two types of wounds: acute wounds and chronic wounds. Acute wounds take 4-6 weeks to fully heal. When the healing process exceeds 6 weeks without resolution, it becomes a chronic wound. Chronic wounds, such as venous ulcers, diabetic ulcers, or pressure ulcers, represent one of the most significant unmet medical needs in the world today and are a major complication of diabetes, resulting in significant morbidity, loss of productivity, and medical expenditure. Diabetic foot ulcers are an important cause of morbidity and are the most common reason for hospitalization of diabetic patients. Approximately 15% of diabetic patients develop chronic ulcers during their lifetime. Of those requiring amputation of a lower extremity, 70-90% will have had a foot ulceration first [4].

[0004] Diabetic wounds are characterized by elevated levels of matrix metalloproteinases (MMPs), increased proteolytic degradation of ECM components, and inactivation of growth factors, ultimately leading to ECM decay that cannot support healing [5, 6]. Abnormal nitric oxide (NO) production also contributes to the pathogenesis of impaired healing. Some cells, such as keratinocytes, fibroblasts, and macrophages, exhibit dysregulated expression and stress responses to many growth factors and cytokines. As a result, these wounds are generally unresponsive to most therapies. For these reasons, an aggressive strategy for intervention can be most beneficial, which can restore the decaying extracellular microenvironment in diabetic wounds in multiple ways that approximate the composition of normal ECM. The regulatory pathways consist of a complex network, making it difficult to design compensatory regulation required for wound repair. Therefore, an aggressive healing strategy that replaces missing or dysregulated extracellular matrix (ECM) components can be most beneficial.

[0005] Prior Art Background

[0006] Over the past decade, vascular endothelial growth factor (VEGF) has been extensively studied and has been reported to have pleiotropic functions in the central nervous system (CNS) and its supporting physiological environment. The functions of VEGF are well known, such as angiogenesis, enhanced vascular permeability, and gliosis, as well as more recently recognized functions such as neuroprotection and even neurogenesis. Recently, the neurogenesis function has received widespread attention, and multiple research groups have participated in the challenge to elucidate this activity. In line with this trend, our understanding of VEGF receptors has increased, and in the current work in progress, certain suggestions about the mechanisms of neuroprotection are being made, although at times researchers can only deal with a very small percentage of VEGF signaling. Neuroplastin (NP), along with flt-1 (VEGF receptor 1) and flk-1 (VEGF receptor 2), is often described as participating in the neuroprotective effects of VEGF. The direct and indirect neuroprotective effects of VEGF are discussed in the context of the latest emerging insights into the biological mechanisms of VEGF and the interacting molecules closely related to it, including various signaling pathways and the neurogenesis induced by this factor [7].

[0007] Leukocytes, particularly neutrophils and macrophages, persist in the surrounding tissue and secrete various proteases, including matrix metalloproteinases (MMPs) and serine proteases [8]. Over-accumulation of these enzymes can interfere with matrix remodeling [9]. Agents that inhibit proteases are thought to be beneficial for wound healing

[10] . Another feature of some chronic wounds is a reduction or absence of angiogenesis, which prevents nutrients from reaching the newly formed tissue

[11] . In the prior art, initial treatments for improving wound healing in chronic wounds include eschar debridement, antibiotic therapy where appropriate, and treatment with regular dressings [2]. Other dressings can also be used, such as hydrogels, hydrocolloids, or alginate. Venous ulcers are treated with compression therapy, while arterial ulcers or diabetic ulcers require regular dressing changes. Alleviating pressure at the site of injury is used to promote healing of pressure sores. Some other physical devices, such as laser therapy, hyperbaric oxygen therapy, and electrical stimulation for arterial ulcers, are also used to promote wound healing [2, 12, 13].

[0008] For wounds that do not respond to such interventions, the use of tissue engineered skin, such as Dermagraft or Apligraf, is an option. This therapy prevents bacterial infection and allows the wound to heal through normal repair processes [14, 15]. The use of such skin substitutes to accelerate wound healing depends on whether existing blood vessels in the existing wound can be supplied. Another approach to wound healing involves the administration of growth factors / cytokines, which have been shown in certain animal models to accelerate cell proliferation in vitro to promote wound healing. These include IL-1, platelet-derived growth factor (PDGF), EGF, VEGF, TGF-β, and bFGF [2]. Procure (Therapeutic Technologies) is an autologous platelet release containing at least five growth factors that help to form granulation tissue and re-epithelialization. This autologous growth factor cocktail has achieved some success in human subjects with ulcerative limb lesions

[16] . However, overall, the results of most clinical trials using growth factors / cytokines have been disappointing. For example, EGF failed to heal venous stasis ulcers, and IL-1 was not effective in treating pressure sores [2]. Results with bFGF were similar

[17] . The reasons for the lack of efficacy are not certain, but can be related to the multifactorial effects of growth factors / cytokines, some of which are not desirable for healing.

[0009] Jackson’s patent

[18] utilizes activated protein C (APC) to aid in wound healing. According to the patent, APC can also act as an anti-inflammatory agent and can directly activate the protease, gelatinase A [18, 19].

[0010] Many different cell types, including smooth muscle cells, fibroblasts, and endothelial cells, can secrete gelatinase A. Gelatinase A plays a crucial role in physiological remodeling and angiogenesis by degrading collagen in the basement membrane

[20] and allowing cell invasion of the substrate. In addition, APC is also able to promote endothelial cell regeneration after in vitro injury, stimulate re-epithelialization, fibroblast invasion, and angiogenesis in chicken embryos, and enhance wound healing in a rat injury model. In combination with the above clotting inhibition, anti-inflammatory, and gelatinase A activation functions, APC, functional fragments thereof, and precursors of APC (i.e., protein C) are suitable for treating wounds, particularly slow-healing wounds.

[0011] Another patent

[22] describes a topical composition for treating skin wounds using colostrum. Growth factors, immune factors, enzymes, and micronutrients and macronutrients are biologically active in colostrum and are associated with wound healing. The growth factors mentioned in this patent include epidermal, fibroblast insulin-like I & II, transforming alpha & beta, and platelet-derived nerve; immune factors include immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, and lactalbumin; enzymes include lysozyme, peroxidase, proteases, and xanthine oxidase; and micronutrients and macronutrients include carbohydrates, amino acids, vitamins, and minerals.

[0012] A patent

[23] describes human plasma fibronectin. In this patent, the formulation allows for slow release of fibronectin at the wound site and increased contact time with the wound site, resulting in effective absorption. Fibronectin is involved in all stages of wound healing. Some of the biological activities of fibronectin associated with wound healing include cell recruitment, opsonization of particulate debris, and promotion of wound contraction

[24] . In addition, topically applied fibronectin is able to increase the rate of wound healing (e.g., corneal wounds [25, 26] and leg ulcers

[27] ). This patent focuses on gel and cream formulations.

[0013] A patent

[28] describes the topical or transdermal delivery of compositions comprising nitric oxide and / or peptides, such as thyrotropin-releasing hormone (TRH) and / or GnRH (gonadotropin-releasing hormone), to promote wound healing.

[0014] Recombinant platelet-derived growth factor-BB (PDGF-BB) (becaplermin) is a growth factor approved by the U.S. Food and Drug Administration for the treatment of diabetic foot ulcers, which functions as a topical cream. PDGF-BB is a potent mitogen and chemotactic agent for stromal cells and can increase wound vascularization by stimulating angiogenesis.

[0015] One U.S. patent

[29] utilizes Angiopoietin-like protein 4 (ANGPTL4) to assist in wound repair. ANGPTL4 has been shown to act on angiogenesis and vascular permeability under certain circumstances [30-32]. A deficiency in ANGPTL4 in mice results in delayed re-epithelialization of wounds, decreased matrix protein expression, increased inflammation, and impaired wound-related angiogenesis [33, 34].

[0016] Particular areas of wound healing include neuroprotection and neuroregeneration of damaged axons and neural tissue. When a mammalian peripheral nervous system (PNS) axon is severely damaged (i.e., a crush injury or a transection injury), a variety of phenomena can occur. First, if left unregulated, the distal stump almost always degenerates (Wallerian degeneration), a process associated with a chromatinolytic change in the perikaryon proximal to the degeneration. If the damage to the nerve is severe enough, the proximal portion of the axon will degenerate, followed by the perikaryon and (usually) death. If the damage to the nerve is less severe and left unregulated, a number of biochemical changes occur in the remaining proximal portion. These proximal changes involve a complex series of reactions by the cell body to the injury, which appear to prepare the intact portion for regeneration. Such changes include alterations in axonal transport properties, protein processing, and nucleic acid synthesis. A regenerative growth cone is observed at the damaged proximal stump, and the axon will regenerate from the proximal stump toward the original target area.

[0017] Such damage can trigger collateral sprouting in neighboring axons that were not severely damaged and did not undergo Wallerian degeneration. The newly grown neurites can use the degenerating distal segment as a guide to denervated target areas. It is hypothesized that reactive Schwann cells provide diffusible growth-promoting factors and provide a suitable growth surface established by the plasma membrane and / or basal lamina or extracellular matrix, providing a cross-bridging means for target regeneration. These morphological and biochemical changes depend largely on several factors, including the severity and location of the injury in terms of proximity to the perikaryon, the size of the damaged axon, and the species involved. For example, the peripheral nervous system (PNS) axons of higher vertebrates have a low capacity for effective regeneration.

[0018] In the central nervous system (CNS), unassisted regeneration is usually aborted rapidly by the substrate, resulting in complete degeneration of the proximal stump and perikaryon.

[0019] Nutrients from nearby tissues can have greater trophic potential than the intended target tissue. Thus, in the prior art, the length of the gap of the injury has been shown to be a major factor in determining the success of functional regeneration. It has been revealed in the literature that this lack of target specificity is the underlying cause of the frequent formation of neuromas and improper contact with other tissues. Thus, a significant improvement is observed after nerve anastomosis. The method of repairing peripheral nerve damage in current neurosurgery is to simply anastomose the cut ends of the nerve.

[0020] Numerous studies have been conducted to determine nerve and / or axon regeneration after neuronal tissue injury. In the adult central nervous system, axons that experience traumatic injury generally do not regenerate spontaneously. Injury to the peripheral nervous system results in neuropathy, which leads to weakness and paralysis, sensory maladies or loss, uncomfortable and painful neuropathies, and impaired autonomic function

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

[0021] Background of existing compositions and formulations

[0022] Gel formulations comprise a water-soluble, pharmaceutically acceptable polymer prepared from an effective amount of fibronectin. Examples of such compounds include: vinyl polymers, such as polyacrylic acid; polyoxyethylene-polyoxypropylene block copolymers, such as poloxamer; and cellulose derivatives, such as hydroxypropyl cellulose (HPC). The polymer has a viscosity value of 50,000 to 1,000,000 cps at room temperature. Cream formulations are prepared from a commercially available cream base, namely, Schering base (Schering Canada Inc., Point-Claire, Quebec) having a viscosity value of 60,000 to 80,000 cps at room temperature. The sustained release system of the gel formulations of the present application allows for slow release of fibronectin to the wound site. This property of the formulations allows for less frequent application to the wound, thereby causing less interference with the healing process. Such formulations maintain fibronectin delivery for up to 24 hours. Preferred embodiments include a twice daily treatment regimen, based on kinetic data.

[0023] One patent

[36] describes a wound healing composition comprising an aqueous mineral oil emulsion capable of easy spreading and film formation, which forms a stable, coherent layer when applied to a moist skin or mucosal surface.

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

[0025] The objective of wound repair includes accelerating wound closure with minimal scarring.

[0026] A number of studies have been performed to investigate the efficacy of epidermal growth factor (EGF) on wound healing, with mixed results. Thornton et al. (1982) applied EGF topically to scalded areas in rats

[37] . A marginal healing advantage over controls was observed. Niall et al. (1982) reported an enhancement of wound healing in mice after topical application of EGF

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

[39] . They reported that it increased the rate of epithelialization of split-thickness wounds in vivo. In a fourth paper, Buckley et al. (1985) investigated the effect of EGF on slow release from a subcutaneously implanted sponge in rats

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

[0027] Lawrence et al. (1986) and Sporn et al. (1983) showed that the application of transforming growth factor (TGF) accelerated wound healing in rats [41, 42], and according to Lawrence et al. (1986) there was further enhancement with the combined use of TGF, EGF and platelet-derived growth factor (PDGF)

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

[43] .

[0028] More recently, a patent

[44] has described IGF, insulin-like growth factor-I (IGF-I) (also known as somatomedin C) and insulin-like growth factor-II (IGF-II) has generated interest. Froesch et al. (1985) reported that the main role of IGFs is to stimulate growth and differentiation of cells of germ layer origin

[45] . They further reported that persistently high levels of bound IGFs in serum can perform a variety of functions: replacement of apoptotic cells, repair mechanisms, matrix synthesis and possibly cell persistence stabilization, preventing their transformation and dedifferentiation

[45] .

[0029] Prior art background to nerve regeneration

[0030] In cases where the damage and degeneration are so extensive that the distance between the remaining proximal and distal stumps is too great for simple anastomosis, an alternative solution is to employ a structure or "bridge" that spans the length of the gap from the cut proximal stump to the distal part of the nerve or target tissue itself. In animal studies, various materials have been used to bridge the gap, including peripheral nerve grafts, mesothelial chambers, microporous and silicone tubes.

[0031] Artificial production and commercially available "nerve cuffs" or "nerve conduits" are of particular interest, which can be implanted between and extend between stumps. Nerve conduits are typically made of silicon or bioabsorbable materials. Nerve conduits can be filled or coated with a growth-supporting matrix, such as laminin, which promotes nerve growth over a greater distance than an unmodified nerve conduit alone.

[0032] A study by Want T et al. showed that branched-chain amino acids have beneficial effects on neuronal survival and axonal regeneration of retinal ganglion cells after transection of the optic nerve through the mammalian target of rapamycin (mTOR) pathway

[46] . Nucleic acid therapy can improve regeneration by enhancing the intrinsic growth ability of neurons and overcoming the inhibitory environment that prevents neurite outgrowth. These nucleic acids modulate gene expression by overexpressing neuronal growth factors or silencing growth inhibitory molecules

[47] . Another study measured neuronal regeneration by studying the olfactory nerve. Dysfunction of this nerve leads to loss of smell. This study combined the delivery of two growth factors, namely vascular endothelial growth factor and platelet-derived growth factor, to increase the number of mature olfactory neurons

[48] . In addition, the neuroprotective activity of curcumin was studied. The effect of local and sustained treatment with low doses of curcumin on nerve regeneration after crush of the sciatic nerve in rats was investigated. Curcumin treatment increased the expression of dense myelin proteins, myelin thickness, and increased motor and sensory nerve conduction velocities. In addition, curcumin treatment reduced the production of reactive oxygen species (ROS), especially by macrophages, lipid peroxidation, and increased expression of the transcription factor Nrf2. This antioxidant capacity can have contributed to the beneficial effects of curcumin after SNC injury

[49] .

[0033] Advanced conduit design and manufacturing techniques have made it possible to manufacture autograft-like structures in the NGC with incredible precision. To this end, strategies involving biopolymers, cells, growth factors, and physical cues have been developed over the past few decades and have facilitated the development of various NGC, from simple hollow tubes to complex conduits containing one or more guidance cues

[35] . In one study, the use of a nerve conduit and stem cells was combined to enhance recovery of the recurrent laryngeal nerve after repeated insults. The results revealed that a laminin-chitosan-PLGA nerve conduit combined with Schwann cells and neural stem cells was able to promote nerve regeneration (P < 0.05) and that its effect was superior to that of an autograft (P < 0.05)

[50] .

[0034] Non-pharmacological agents have been used for nerve regeneration, such as electrical stimulation. One study evaluated the regenerative effects of electrodes with different contacts implanted in excised sciatic nerves. This approach showed an increase in sciatic functional index, compound muscle action potential amplitude, motor nerve conduction velocity, and a reduction in muscle atrophy

[51] . Another study on peripheral nerve injury showed that electrical stimulation combined with neural crest stem cells significantly enhanced nerve regeneration and repair after injury. These findings were comparable to autograft therapy

[52] .

[0035] After injury, an axon can re-engage with its target tissue using one of a variety of different strategies. It first needs to initiate regrowth, which can originate from the tip of the cut axon end still attached to the cell body, from a branch extending out of the fragment, or from a new axon from the body cell. To reach its target, the axon can navigate along the entire length of its original path, or use a different ectopic route. As extensive regrowth can be required, the regrowing axon can instead establish functional connections with other neurons in the vicinity, take a similar route, or establish functional connections with new neurons after cell migration to the damaged area, thus shortening the growth requirement. As customary, peripheral nerve injuries causing major gaps between segments are repaired with autologous nerve grafts

[53] . An alternative approach, especially for severe injuries, is end-to-side neurorrhaphy, in which the transected distal nerve stump is joined to the trunk of an adjacent intact donor nerve

[54] . This technique has been used for various peripheral nerve injuries, but the results are mixed and its use is limited due to the lack of randomized clinical trials

[54] . Combining this approach with the use of endothelin B receptor agonists, such as sarafotoxin (IRL-1620), can help functional recovery and expand the clinical usefulness of nerve suture.

[0036] Growth factors (GF) (e.g., epidermal, fibroblast, insulin-like I and II, transforming O & B, platelet-derived, nerve), immune factors (e.g., immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, lactalbumin), enzymes (e.g., lysozyme, peroxidase, proteases, xanthine oxidase), micronutrients and macronutrients (e.g., carbohydrates, amino acids, vitamins, minerals) have also been proposed for treating wounds and repairing damaged nerves.

[0037] Background of endothelin analogs related to wound healing

[0038] There are many compositions that can enable tissue structure healing. Endothelin has been reported to enhance many of the cascading processes that are effective for 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 extracellular matrix (ECM) (e.g., collagen type I and fibronectin) secretion in vitro. The results indicated that ET-1 can promote proliferation, migration, secretion, and secretion of ECM through protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) signaling pathways. This revealed that ET-1 can play a crucial role in the development of proliferative vitreoretinopathy (PVR). PVR in turn represents an exaggerated wound healing response via ECM molecules

[55] .

[0039] Endothelin has also been studied in liver wound healing. For example, in a study by Khimji et al. (2011) it was found that the amount of ET-1 production increased during liver injury and that the cellular source of ET-1 shifted from endothelial cells to stellate cells. This established a feedback loop that emphasized further activation, stellate cell proliferation, and ECM protein production

[56] .

[0040] Another study by Lagares et al. (2010) indicated that endothelin 1 contributes to the action of transforming growth factor B1 (TGFB1) on wound repair and skin fibrosis. TGFB1 induced ET-1 expression in human dermal fibroblasts through Smad-dependent and activin 1 / JNK-dependent signaling. The ability of TGFB1 to induce pro-fibrotic gene expression was dependent on ET-1. In mouse skin, adenovirus-mediated overexpression of TGFB1 and ET-1 was associated with accelerated wound closure, increased fibrogenesis, and scarring

[57] . According to this study, ET-1 receptor antagonists, such as bosentan, can represent useful therapeutic tools to treat diseases associated with excessive scarring and fibrosis

[57] . Another study evaluated the role of ET-1 in the wound healing process, which indicated that skin wound healing was accelerated when ET-1 was not attenuated via bleomycin. ET-1 mice exhibited earlier granulation tissue and re-epithelialization

[58] .

[0041] Endothelin is involved in neurogenesis and neuroprotection. According to a study, ischemic stroke-induced rats given an endothelin B receptor agonist were compared to a control group that was not given the drug. In the rats in the endothelin B receptor agonist group, the number of blood vessels labeled with vascular endothelial growth factor increased, the number of neurons was better preserved, and the expression of nerve growth factor increased [59-62]. SUMMARY

[0042] Aspects of the claimed embodiments include a novel composition comprising an endothelin B agonist for enhancing 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. Specifically, the present application aims to use this composition to treat patients with acute or chronic wounds, including diabetic foot ulcers. Furthermore, the present application aims to provide a composition that aids in the regeneration of nerve tissue after acute or chronic nerve injury, such as transection of a nerve that occurs during surgery. In preferred embodiments, the endothelin B agonist can have various compositions, including but not limited to bioactive factors, stem cells, proteins, and elements for enhancing tissue recovery. Topical drug delivery of the preferred embodiments can be local or placed in deep tissue intraoperatively or injected. Systemic delivery can be an option for diffuse tissue injury cases. These delivery systems include intravenous, intramuscular, and oral forms. BRIEF DESCRIPTION OF DRAWINGS

[0043] These and other features, aspects, and advantages of the present application will become better understood with reference to the following description, appended claims, and accompanying drawings where:

[0044] Figure 1 A chromatogram of IRL-1620 is shown (retention time 5.093).

[0045] Figure 2 An image of the cross-linking stage of alginate hydrogel films prior to lyophilization is shown.

[0046] Figure 3 An image of fully cross-linked alginate hydrogel films prior to lyophilization is shown.

[0047] Figure 4 Disinfection using UV radiation in laminar flow is shown.

[0048] Figure 5 An image of lyophilized hydrogel alginate films is shown.

[0049] Figure 6 Fourier transform infrared spectra (FTIR) of hydrogel films is shown.

[0050] Figure 7 FTIR of sodium alginate is shown.

[0051] Figure 8 FTIR of IRL-1620 is shown.

[0052] Figure 9Scanning electron microscopy (SEM) images showing surface view of sodium alginate hydrogel films containing (a) sodium alginate-1%, (b) sodium alginate-1.25%, c) sodium alginate-1.5%, (d) sodium alginate-2%.

[0053] Figure 10 Light transmission properties of hydrogel films are shown.

[0054] Figure 11 In vitro release pattern of IRL-1620 from the formulation through goat skin is shown up to 26 hours.

[0055] Figure 12 In vitro release pattern of IRL-1620 from the formulation through goat skin is shown up to 5 hours.

[0056] Figure 13 Cumulative % release pattern of IRL-1620 from the formulation through goat skin is shown.

[0057] Figure 14 % Assay of stability samples of the developed formulation under refrigerated (2-8 °C) and deep frozen (-20 °C) conditions is shown.

[0058] Figure 15 Chromatogram of IRL-1620 (Retention time 5.093) is shown.

[0059] Figure 16 Microscopic view of niosomes F2 and F3 is shown.

[0060] Figure 17 Encapsulation efficiency of different formulations is shown.

[0061] Figure 18 FTIR spectrum of IRL-1620 is shown.

[0062] Figure 19 FTIR spectrum of excipient (soy lecithin) is shown.

[0063] Figure 20 FTIR spectrum of niosomes (F3) is shown.

[0064] Figure 21 Particle size distribution graph is shown.

[0065] Figure 22 Zeta potential distribution peak is shown.

[0066] Figure 23 Niosome gel G-3 (for in vitro release and wound healing studies in rats) is shown.

[0067] Figure 24 In vitro release pattern of IRL-1620 through goat skin in formulation G-3 is shown.

[0068] Figure 25 Cumulative % release pattern of IRL-1620 through goat skin in formulation G-3 is shown.

[0069] Figure 26 % assay of stability samples of developed formulations at room temperature and refrigerated conditions is shown.

[0070] Figure 27A , Figure 27B , Figure 27C , Figure 27D , Figure 27E , Figure 27F , Figure 27G , Figure 27H : Effect of different treatments on inflammation, angiogenesis and fibroblast proliferation in corresponding groups of excision model is shown.

[0071] Figure 28A , Figure 28B , Figure 28C , Figure 28D , Figure 28E , Figure 28F , Figure 28G , Figure 28H : Effect of different treatments on collagen deposition in corresponding groups of excision model is shown.

[0072] Figure 29A , Figure 29B , Figure 29C , Figure 29D , Figure 29E , Figure 29F , Figure 29G , Figure 29H : Effect of different treatments on VEGF expression in corresponding groups of excision model is shown.

[0073] Figure 30A , Figure 30B , Figure 30C , Figure 30D , Figure 30E , Figure 30F , Figure 30G , Figure 30H : Effect of different treatments on vWF expression in corresponding groups of excision model is shown.

[0074] Figure 31 : Effect of different treatments on wound healing in different groups is shown.

[0075] Figure 32A , Figure 32B , Figure 32C , Figure 32D , Figure 32E, Figure 32F , Figure 32G , Figure 32H : shows the effect of different treatments on inflammation, angiogenesis and fibroblast proliferation in the corresponding groups of the excision model.

[0076] Figure 33A , Figure 33B , Figure 33C , Figure 33D , Figure 33E , Figure 33F , Figure 33G , Figure 33H : shows collagen deposition in different groups of the incision model.

[0077] Figure 34A , Figure 34B , Figure 34C , Figure 34D , Figure 34E , Figure 34F , Figure 34G , Figure 34H : shows VEGF expression in different groups of the incision model.

[0078] Figure 35A , Figure 35B , Figure 35C , Figure 35D , Figure 35E , Figure 35F , Figure 35G , Figure 35H : shows vWF in different groups of the incision model. DETAILED DESCRIPTION

[0079] A method for enhancing wound healing in tissue, including superficial tissue structures and deep tissue structures, including but not limited to epithelium, subcutaneous tissue, muscle tissue, fascia, bone, and neurovascular tissue is disclosed. Specifically, the present application aims to treat patients with acute or chronic wounds, including diabetic foot ulcers, using the composition. In addition, the present application aims to provide a composition that aids in the regeneration of nerve tissue following acute or chronic nerve injury, such as a nerve transection that occurs during surgery. The endothelin B receptor plays an important role in neuroprotection and nerve regeneration, while also enhancing the physiological wound healing cascade. The present application involves the use of endothelin B receptor analogs such as N-succinyl-[Glu9,Ala11,15]endothelin 1 (sarafotoxin, IRL-1620), BQ-3020, [Ala1,3,11,15]-endothelin, anguirus S6c, and endothelin 3 for tissue healing. Sarafotoxin (IRL-1620) enhances blood flow to the brain, increases angiogenesis, neurogenesis, reduces apoptosis and mitochondrial fission. IRL-1620 induces differentiation of neural progenitor cells into mature neurons. The anti-apoptotic, anti-inflammatory, and anti-oxidative activities of IRL-1620 limit cell death, and it also promotes the formation of new neurons (neurogenesis) and new blood vessels (angiogenesis), thereby enhancing the healing of wounds and nerve regeneration.

[0080] Definitions

[0081] As used herein, the term "amount sufficient to" refers to an amount that is capable of achieving an intended effect, e.g., in increasing fibroblast cells in a wound bed or in improving conduction through a neural pathway. Such an amount can be determined by various assays known in the art based on the intended effect. As used herein, the term "application" or "administration" refers to all means of introducing a specified agent, composition, or force into a specified area or subject. "Administration" or "application" can be carried out continuously or intermittently throughout treatment in one dose. Methods of determining the most effective administration means and dosage are known to those of skill in the art and will vary based on the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage amounts and methods of administering agents are known in the art. Routes of administration can also be determined, methods of determining the most effective route of administration are known to those of skill in the art and will vary based on the composition used for therapy, the purpose of the therapy, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include oral administration, nasal administration, inhalation, injection, and topical administration. Administration can be used in industrial as well as therapeutic applications. As used herein, the term "biodegradable" is used herein to describe a substance, e.g., a polymer, composition, and formulation, that is expected to degrade during use. A biodegradable substance can also be "biocompatible," i.e., not harmful to living tissue. Non-limiting exemplary biodegradable substances include poly lactic acid (PLA) and poly(lactic-co-glycolic) acid (PLGA), optionally pegylated.

[0082] The term "nerve tissue" refers to tissue that improves signaling of motor and sensory neural pathways. This can include neurons, nerve cells, glial cells, axons, astrocytes, microglia, ependymal cells, oligodendrocytes, enteric glial cells, satellite cells, and Schwann cells. Nerve tissue also includes certain portions of the central nervous system, including the brain and spinal cord, as well as the peripheral nervous system that regulates and controls body functions and activities.

[0083] As used herein, the term "tissue" refers to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism.

[0084] As used herein, the term "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired effect. In the context of therapeutic applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to the pharmaceutical composition. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of in vitro applications, the effective amount will depend in some embodiments on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods used. The skilled artisan will be able to determine effective amounts based on these and other considerations. The effective amount can include one or more administrations of the composition, depending on the embodiment. The dosage range for N-succinyl-[Glu9, Ala11, 15]endothelin 1 (IRL-1620, sarafin) can be from 0.00001 to about 1 mg, and can be administered one or more times over a day, weeks, or months.

[0085] As used herein, the term "treating" or "treatment" includes preventing a subject susceptible of or afflicted with a disease, disorder, and / or condition from developing the disease, disorder, or condition; inhibiting the disease, disorder, or condition, e.g., impeding its progress; and relieving 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 the particular disease or condition.

[0086] Composition

[0087] The present application relates to a composition or agent that is capable of regenerating lost or damaged tissue. The basic composition comprises an endothelin B receptor agonist, with or without stem cells. In some embodiments, the basic composition can not comprise stem cells.

[0088] In some embodiments, the base composition can 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, neurotrophins selected from the group consisting of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, nerve growth factor, vascular endothelial growth factor, and ciliary neurotrophic factor, insulin-like growth factor, Netrinl, thyroid hormone, hepatocyte growth factor, luteolin, tenascin, epidermal growth factor, transforming growth factor-beta, human plasma fibronectin, platelet-derived growth factor, and basic fibroblast growth factor. Additionally, embodiments can include Prochen (Therapeutic Technologies), an autologous platelet release containing at least five growth factors that aid in the formation of granulation tissue and re-epithelialization. This autologous growth factor cocktail has achieved some success in human subjects with ulcerative limb lesions.

[0089] In some embodiments, the base composition can be combined with chronic wound healing strategies including, but not limited to, eschar debridement techniques, antibiotic therapy, compression therapy, laser therapy, hyperbaric oxygen therapy, and electrical stimulation. Dressings for such wounds can include hydrogels, hydrocolloids, or alginates, and can be part of embodiments.

[0090] In some embodiments, the base composition can be combined with compounds known to aid in wound healing, including activated protein C, which promotes the regeneration of endothelial cells. Another compound includes colostrum, which contains growth factors, immunological factors, enzymes, and micro- and macro-nutrients that are biologically active and relevant to wound healing. Angiopoietin-like protein 4 (ANGPTL4) is another compound that aids in wound repair. ANGPTL4 has been shown to play a role in angiogenesis and vascular permeability under certain circumstances.

[0091] In some embodiments, the base composition can be combined with compounds that have been shown to promote neuronal survival, recovery, and regeneration, including branched chain amino acids, nucleic acids that overexpress neuronal growth factors, and curcumin.

[0092] In some embodiments, the base composition can be combined with immunological factors, including immunoglobulins, lactoferrin, cytokines, proline-rich polypeptides, and lactoperoxidase.

[0093] In some embodiments, the base composition can be combined with hormones, including thyrotropin-releasing hormone and gonadotropin-releasing hormone.

[0094] In some embodiments, the base composition can be combined with enzymes and / or micronutrients and macronutrients including lysozyme, peroxidase, protease, xanthine oxidase, carbohydrates, amino acids, vitamins, and minerals.

[0095] In some embodiments, the base composition can be combined with compounds that promote degradation processes that promote wound healing, such as gelatinase A.

[0096] In some embodiments, stem cells used in the base composition can include, but are not limited to, hematopoietic stem cells, embryonic stem cells, and stem cells from platelet-rich plasma. In some embodiments, stem cells can also be genetically modified as a vehicle to produce factors that promote regeneration of damaged tissue, including neural tissue. Finally, in some embodiments, stem cells can be transplanted into damaged tissue prior to administration of endothelin B receptor agonists.

[0097] In some embodiments, the base composition can be combined with tissue-engineered skin such as Dermagraft or Apligraf. This therapy can prevent bacterial infection and allow the wound an opportunity to heal through normal repair processes.

[0098] Preparation of preferred embodiment of a stock solution of sovaspeptin (IRL-1620)

[0099] Bulk drug substance

[0100] IRL-1620 (98.5% purity) was manufactured and sold by American Peptide Company, Inc., 1271 Chestnut Avenue, Suite Vista, CA 92081, USA. Sodium alginate, glycerol, calcium chloride, and all other excipients and solvents were purchased from Merck.

[0101] Pharmaceutical analysis

[0102] Analytical method for IRL-1620 (Sovaspeptin)

[0103] IRL-1620 was analyzed using HPLC method. Analysis was performed using gradient HPLC, 1% TFA in water and 0.1% TFA in acetonitrile (55:45) as mobile phase respectively. Fresh mobile phase was prepared every day. The mobile phase was filtered through 0.2 mm membrane filter to remove any particulate matter, mixed and degassed by sonication before use, its stability was observed, there should be no precipitation with time or temperature reduction. The absorbance of IRL-1620 was fixed at 215 nm wavelength. The sensitivity of the detector was set to 0.01 AUFS. The column was equilibrated with mobile phase flowing through the system for at least 60 minutes before injecting the solution. Each solution was injected in triplicate, the relative standard deviation (RSD) based on peak area was required to remain below 1.0%. Details of the chromatographic conditions are as follows:

[0104] Table 1 Chromatographic conditions for IRL-1620 analysis

[0105]

[0106]

[0107] Preparation of IRL-1620 stock solution

[0108] Accurately weigh 1.0 mg of IRL-1620 lyophilized powder and transfer it to a 10 mL volumetric flask. Make up the volume with HPLC water to get a concentration of 100 pg / mL of IRL-1620 (IRL-1620 stock-I). Next, take an aliquot of 100 pi of the above solution (stock-I) into a 10 mL volumetric flask, make up the volume with diluent to get a concentration of 1.0 pg / mL. Label the above solution as IRL-1620 stock-II and store it at -20 °C. Use the stock solution of IRL-1620 for a period of 30 days and store it appropriately at -20 °C.

[0109] Formulation and mode of administration

[0110] Formulations containing the basic composition to promote wound healing and nerve regeneration, with or without stem cells, are referred to as endothelin B receptor agonists.

[0111] In some embodiments, the basic composition to promote wound healing and nerve regeneration can be synthesized in different formulations and / or particles as described below.

[0112] The composition can be formulated in the form of a gel, cream, lotion, paste, solution, solid, adhesive patch with reservoirs (so that the composition can be released slowly) and the like, which can be wiped or sprayed on the affected area in need of regeneration or healing, including skin, subcutaneous tissue, muscle, fascia, blood vessels and nerves.

[0113] In some embodiments, the formulation can comprise a water-soluble gel polymer prepared from an effective amount of fibronectin. Examples of such compounds include: vinyl polymers, such as polyacrylic acid; polyoxyethylene-polyoxypropylene block copolymers, such as poloxamer; and cellulose derivatives, such as hydroxypropyl cellulose (HPC). The polymer has a viscosity value of 50,000 to 1,000,000 cps at room temperature. The cream formulation is prepared from a commercially available cream base, namely Schering base (Schering Canada Inc., Point-Claire, Quebec) having a viscosity value of 60,000 to 80,000 cps at room temperature. The sustained release system of the gel formulation slowly releases fibronectin to the wound site. This property of the formulations allows for less frequent application to the wound, thereby causing less interference with the healing process. Such formulations maintain fibronectin delivery for up to 24 hours. Preferred embodiments, in light of the kinetic data, prefer a twice daily treatment regimen in this case.

[0114] In some embodiments, the delivery modality can comprise an aqueous mineral oil emulsion, which spreads easily and forms a film, and which forms a stable, coherent layer when applied to a moist skin or mucosal surface.

[0115] In some embodiments, the delivery modality can comprise a medicated dissolvable sponge, such as a gel foam. This can be implanted subcutaneously at the wound bed or during surgery, and then the incision defect is closed.

[0116] For specific nerve tissue regeneration, in preferred embodiments, the delivery modality can employ a structure or "bridge" spanning the length of the gap from the proximal nerve stump to the distal portion of the nerve or target tissue itself. In animal studies, various materials for bridging the gap include peripheral nerve grafts, mesothelial chambers, microporous and silicone tubes. The nerve guide tube can be made of silicone or bioabsorbable material. The nerve guide tube can be filled or coated with the preferred composition. The tube can be designed as an autograft-like structure in combination with the preferred composition. Biopolymers, cells, growth factors and physical stimuli can be incorporated into the tube to achieve one or more guidance cues. For example, a nerve conduit can be made of laminin-chitosan-PLGA and replaced with our preferred composition and stem cells to enhance recovery of the damaged nerve.

[0117] 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 can comprise additional additives including, 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 optional superparamagnetic iron oxide (SPION). In some embodiments, the nanoparticles can also be comprised in a solution, suspension, gel or other suitable formulation for its delivery.

[0118] The embodiments can be formulated to facilitate delivery; for example, a second agent that is poorly water soluble (e.g. TIDE) can be encapsulated in an organic shell of ETBRA with or without stem cells loaded with hydrophilic agents that regenerate damaged tissue.

[0119] The preferred embodiments of the present application for topical drug delivery are vesosome gels and thin film hydrogels.

[0120] Thin film hydrogel embodiments

[0121] The hydrogel developed for experimental purposes was a thin film of calcium alginate hydrogel. The steps followed for development were as follows:

[0122] Materials and methods

[0123] Analysis of IRL-1620 in hydrogel samples by HPLC

[0124] About 10 mg of hydrogel sample was weighed in a centrifuge tube. To this tube, 10 ml of phosphate buffer (pH 7.4) was added and sonicated for 30 minutes at 2-8 °C. It was then centrifuged at 5000 rpm at 5 °C and the supernatant was collected. Next, 0.5 ml of the supernatant was taken in a 10 ml volumetric flask and made up to volume with diluent (acetonitrile: water 40:60) (v / v solvent). The sample was analyzed using HPLC method.

[0125] Preparation of alginate-based films

[0126] Alginates films were prepared using solvent casting process (Pereira et al., 2013). Sodium alginate solution (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 (about 2 hours). During the preparation of the alginate solution, the plasticizer (glycerol) was added at a percentage of 15% (w / w) based on the weight of the alginate powder. IRL-1620 was dispersed in 1 ml of water and stirred at 600 rpm in an ice bath. Then, the solution was transferred to an ultrasonic system (30 minutes) under controlled temperature (2-8°C). It was degassed in a refrigerator, 2-8°C, for 12 hours. The alginate pure film containing glycerol was prepared by casting 5 mL of the solution into a Petri dish The film was left for 5 minutes. Next, 2 mL of a 5.0% (w / v) aqueous solution of CaCl2 was poured into the Petri dish and gently stirred to disperse the CaCl2 uniformly and crosslink for 15 minutes. Then, the hydrogel was washed with distilled water, sterilized under UV irradiation in a laminar flow device, and kept deep frozen for 48 hours. The film was lyophilized using a freeze-drier (Allied Frost) until a constant weight was reached (Di Micco et al., 2007).

[0127] Compatibility studies

[0128] The compatibility between IRL-1620 and the excipient sodium alginate was evaluated using FTIR peak matching method (Riaz & Ashraf, 2015). The instrument used was a Bruker FTIR (alpha-1) in the range of 4,000 cm -1 to 650 cm -1 .

[0129] Scanning electron microscope (SEM) studies

[0130] The surface morphology of the samples containing different concentrations of sodium alginate was investigated by scanning electron microscopy (SEM LEO 43 SVP, Cambridge) (Hashem et al., 2013).

[0131] Film thickness

[0132] The film thickness was measured using a vernier caliper micrometer at a precision of 0.001 mm in five different locations of the film. The results were expressed as the mean ± standard deviation (SD) of the measured values.

[0133] Film light transmission and clarity

[0134] The light transmittance and transparency of the films were determined by spectrophotometry according to the method used by Norajit et al. Rectangular samples (10 mm x 35 mm) of the films were cut and placed in a spectrophotometer cell, using air as a reference, their light blocking properties were measured at wavelengths comprised between 200 and 800 nm. The transparency of the films was determined by the following equation.

[0135] Transparency = Abs 600 / x

[0136] where Abs600 represents the absorbance value at 600 nm and x corresponds to the film thickness in millimeters (Norajit & Ryu, 2012).

[0137] Water-solubility

[0138] It is generally accepted that water-solubility and swellability are important properties to characterize biodegradable materials for tissue regeneration and drug delivery applications.

[0139] To determine the water-solubility, the cross-linked samples of the hydrogel alginate films were lyophilized and then immersed in 40 mL of distilled water and stirred at 120 rpm for 24 hours. Then, the samples were taken out of the medium, excess water was removed with filter paper and their wet weight was immediately determined to calculate the water uptake. The samples were then dried in an oven at 37°C until constant weight to determine the dissolved mass.

[0140] The water uptake and water-solubility were determined by the following equations:

[0141] Water uptake (%) = [(Ww - Wd) / Wd] x 100

[0142] Water-solubility (%) = [(Wi - Wf) / Wi] x 100

[0143] 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).

[0144] Swelling properties

[0145] To determine the swelling properties of the hydrogel films, a small rectangular film was cut to a size of 1 x 1 cm and weighed. The film was then placed in 20 ml of phosphate buffered saline (pH 7.4) in a petri dish. After 30 minutes the hydrogel was taken out of the buffer and re-weighed. Thus, the swelling properties were measured as the “% Swelling Index”

[0146] % Swelling Index = [(Wh - Wd) x 100] / Wd

[0147] Wh is the weight of the product after 30 minutes of hydration, and Wd is the weight of the dried product (SWKim et al., 1992).

[0148] Biodegradation research

[0149] Place a small section (2x2 cm) of the membrane in 25 ml of phosphate buffer at room temperature for 7 days. Visually observe the solubility of the membrane.

[0150] In vitro drug release studies

[0151] In vitro dissolution studies of the hydrogel membrane were conducted using goat skin. Fresh goat skin was collected from a local slaughterhouse. It was cleaned and the hair was removed. The permeability of IRL-1620 was assessed using a Franz diffusion cell at 100 rpm in 50 ml of phosphate-buffered saline (pH 7.4) as the dissolution medium, maintained at 37 ± 5 °C. 0.5 ml aliquots were taken at specified time intervals over 48 hours, and the concentration of IRL-1620 was analyzed by HPLC. An equal volume of fresh medium was replaced after each sampling to maintain a constant volume (Thu et al., 2012). The samples were diluted with 0.5 ml of acetonitrile:water (40:60, v / v) and analyzed by HPLC.

[0152] Stability Study

[0153] The stability of hydrogel membrane formulations was evaluated by storing them in sealed glass vials under three different storage conditions (i.e., deep freezing (-20°C), refrigeration (2–8°C), and room temperature) for 45 days (Sabale & Vora, 2012). Samples were taken at different time intervals during the study period, and the membrane percentage was determined by HPLC.

[0154] result

[0155] Drug Analysis

[0156] The developed high-performance liquid chromatography (HPLC) method for quantifying IRL-1620 is simple and sensitive. Separation is performed on a reversed-phase C18 column (250 mm × 4.6 mm), and the column elution is monitored at 215 nm using a UV detector. The method was found to be precise, accurate, and specific. It has been successfully applied to studies of encapsulation efficiency, drug release, and stability. Figure 2 ).

[0157] Preparation of hydrogel membranes

[0158] Due to the formation of calcium alginate, a transparent film can be obtained by adding calcium chloride solution. Figure 2 The membrane was allowed to harden for 15 minutes to obtain a hardened, homogeneous, continuous, flexible hydrogel membrane.Figure 3 These membranes were then subjected to deep freezing and lyophilization. After lyophilization, a white hydrogel membrane was obtained. Figure 5 It has a smooth surface, is flexible, and is not easily broken.

[0159] Compatibility study

[0160] In the drug-polymer mixture, the absence of IR spectral peaks or the disappearance of IR spectral peaks confirms that there is no chemical interaction between the drug and sodium alginate. Figure 6-8 As shown.

[0161] Surface morphology study using SEM

[0162] The surface morphology of the lyophilized hydrogel membranes was studied using SEM. The surface morphology of membranes with different concentrations of sodium alginate was also investigated. Figure 9 The best shape view is (). Figure 9 (d) Therefore, it was used for further research. The 1.5% sodium alginate film had a smooth and uniform texture (depicting the fine cross-linking of the alginate moieties). Concentrations of 1% and 1.25% produced fragile films with many cracks. Higher concentrations (2%) of sodium alginate showed the formation of rough films.

[0163] film thickness

[0164] The developed hydrogel membranes exhibited a smooth surface, high stretchability, and a thickness ranging from 110 to 120 μm (Table 2). Membrane thickness affects its in vivo dissolution in the wound area; therefore, a low concentration of sodium alginate (Tokarev & Minko, 2010) was chosen. This was also sufficient to maintain the membrane's mechanical strength and reduce its brittleness.

[0165] Table 2 Membrane characterization results

[0166] Parameters Results Film thickness 110-120 μm Film transparency 1.80 Water solubility at 37°C (% by mass of dissolved) 8.32 Water absorption rate (%) after 24 hours 146.81

[0167] Membrane transmittance and transparency

[0168] Light transmittance and transparency are important parameters to be evaluated in determining the quality and effectiveness of membranes in local wound healing (Norajit & Ryu, 2012). Low concentrations of IRL-1620 do not affect membrane transparency.

[0169] Water-soluble

[0170] The water solubility and swelling properties of the hydrogel were investigated to evaluate the membrane's performance when it is in permanent contact with biological fluids or tissues. The water solubility at 24 hours was approximately 8.32% (Table 2). Therefore, the membrane can be placed in open wounds for a longer period of time.

[0171] Swelling properties

[0172] The swelling properties of the hydrogel film were 146.8%; this high swelling capacity of the hydrogel can be attributed to the hydrophilic functional groups such as NH2- and COO- from the alginate polymer

[63] . Thus, the developed hydrogel film can be used as a wound healing agent with high water solution absorption capacity. Further, it also helps in the absorption of wound exudates.

[0173] Biodegradation study

[0174] The samples were observed for solubility every day. The film swelled immediately and the size of the film gradually decreased every day. Finally, a homogenous solution was formed on the 7th day of the study. Thus, the degradation and release of alginate from the film in the phosphate buffer took 7 days.

[0175] In vitro drug release study

[0176] In the in vitro study of alginate hydrogel film, the formulation was analyzed by goat skin in PBS (pH 7.4) using Franz diffusion cell and the results are shown in Figure 11 & Figure 12 From the data, it was observed that the in vitro drug release of IRL-1620 from the hydrogel film formulation increased sharply within 5 hours, followed by a stable concentration for about 24 hours, where the release decreased sharply after 26 hours. The cumulative release of IRL-11620 from the formulation was 86.54% after 24 hours. Figure 13

[0177] Stability study

[0178] The alginate film formulation was kept under different storage conditions for 45 days for the stability study. The samples kept at room temperature showed changes in their physical appearance. The color of the film changed after 15 days of storage; thus, it can be considered that the film is not stable at room temperature. The samples kept in the refrigerator and under deep freezing showed physical stability, and thus, IRL-1620 was determined using the HPLC method, and the results are shown in Figure 14

[0179] From the results, it was observed that the drug content decreased at 2-8°C, whereas no significant change was observed in the case of deep freezing (-20°C). Thus, at the end of the study, it was found that the developed hydrogel formulation is stable under deep freezing (-20°C).

[0180] Conclusion

[0181] ​​A hydrogel dressing based on alginate was successfully developed by solvent casting method using glycerol as a plasticizer and calcium chloride as a crosslinking agent. The hydrogel film with 1.5% sodium alginate concentration had a smooth surface and good mechanical strength. The swelling factor and water solubility of the hydrogel proved its suitability for use in wound healing for a longer period. The release of IRL-1620 was up to 24 hours; however, the alginate film remained in contact with the tissue, which could continue to heal the wound by maintaining moisture in the wound area. The formulation was stable at -20°C.

[0182] Vesiculome gel embodiments

[0183] Another alternative of our preferred embodiment is the vesiculome gel with hydrophilic and hydrophobic nature. They allow slow release of the entrapped drug and penetration through the epithelial layer due to the presence of cholesterol

[64] .

[0184] Evaluation of IRL-1620 in vesiculome samples by HPLC analysis

[0185] About 1.0 mg of vesiculome was accurately weighed in a 10 ml volumetric flask. 0.5 ml of butanol was added and shaken to wet the vesiculome completely. It will disrupt the cholesterol and soy lecithin membrane. Diluent solvent was added and made up to 10 ml. The sample was analyzed using HPLC method.

[0186] Estimation of IRL-1620 content in vesiculome gel samples by HPLC

[0187] About 100 mg of vesiculome gel was accurately weighed, dispersed in 10 ml of phosphate buffer (pH 6.8) and centrifuged at about 3000 rpm. The supernatant was removed, the settled residue was treated with 0.5 ml of butanol and the volume was made up to 10 ml using acetonitrile: water (40:60, v / v). The sample was analyzed using HPLC method.

[0188] Preparation of vesiculome

[0189] IRL-1620 was dissolved in ethanol for the preparation of vesiculome. Vesiculome complexes were prepared using ethanol as reaction medium in the ratio (1 : 1, 1 :2, 1 :3, 1 :4 and 1 :5 drug and soy lecithin); the cholesterol concentration was constant. Soy lecithin and cholesterol were dissolved in 10 ml of dichloromethane (Pando et al., 2013) and 5 ml of ethanol containing IRL-1620 (20 pg / ml) was added dropwise under continuous stirring and sonicated for 15 minutes. The resulting mixture was evaporated under vacuum at 2-8°C. The residue (vesiculome) was kept in a desiccator. The vesiculome was stored at 2-8°C.

[0190] Table 3 Vesiculome composition of IRL-1620

[0191]

[0192]

[0193] Characterization of the niosome complex

[0194] Optical microscopy was used to characterize the complex. The complex was suspended in buffer, a drop was placed on a glass slide and covered with a coverslip. Microscopic view of the complex was observed under 10x10 magnification (Junyaprasert et al., 2008).

[0195] Encapsulation efficiency (EE)

[0196] Ten mg of niosomes were added to 10 ml of phosphate buffer (pH 7.4) and centrifuged at 12000 rpm for 45 min using a Remi centrifuge to separate the niosomes from the unencapsulated drug. The separated sedimented niosomes were treated with 0.5 ml of butanol and the volume was made up to 10 ml using acetonitrile: water (40:60, v / v). The concentration of IRL-1620 in the supernatant was determined by using HPLC. The percentage of drug encapsulation was calculated by using the following formula (Singh et al., 2011),

[0197] Encapsulation efficiency (%) = (Total drug weight - Unencapsulated drug weight) x 100 Total drug weight Total drug weight - free drug weight

[0198] Compatibility studies

[0199] FTIR peak matching method was used to evaluate the compatibility between IRL-1620 and excipients, soy lecithin and cholesterol (Lopez-Lorente & Mizaikoff, 2016). The instrument used was a Bruker FTIR (alpha-1) in the range of 4,000 cm -1 to 650 cm -1 .

[0200] Particle size analysis

[0201] A magnetic stirring type cell dispersing unit (Malvern Instruments Ltd., UK) was used for moderate stirring to keep the niosomes dispersed during size measurement. The measurement position was 1.05 mm and the polydispersity analysis mode was selected. These conditions allowed accurate measurement of particles in the range of 0.1 nm to 10000 nm

[65] .

[0202] Zeta potential analysis

[0203] ​The significance of zeta potential is that its value is related to the stability of the colloidal dispersion. The zeta potential value indicates the degree of repulsion between vesicles of similar charge in the dispersion. The zeta potential of the vesicle dispersion was also determined using a Malvern instrument

[66] .

[0204] Development of topical gel of vesicles

[0205] A topical gel of vesicles was formulated using hydroxyethyl cellulose (HEC), triethanolamine (TEA), polyethylene glycol 400 (PEG), methyl paraben, and water. Different concentrations of HEC were dispersed in water to determine the appropriate concentration to form a gel. The ideal ratio was found to be about 11% (w / v) of the solution. To disperse the lipophilic vesicles in the HEC gel matrix, TEA was used as a surfactant, and PEG was used as a stabilizer. Various combinations were prepared as shown in Table 4.

[0206] Table 4 Composition of different IRL-1620 vesicle gel formulations

[0207] Formulation Drug Surfactant Stabilizer Gelling agent G-1 IRL-1620 (1) ------- ------ HEC (7.5) + water (66) G-2 IRL-1620 (1) TEA (2.5) ------ HEC (7.5) + water (66) G-3 IRL-1620 (1) TEA (2.5) PEG (23) HEC (7.5) + water (66) G-4 IRL-1620 (1) TEA (2) PEG (23.5) HEC (7.5) + water (66) G-5 IRL-1620 (1) TEA (1.5) PEG (24) HEC (7.5) + water (66)

[0208] Excipient details

[0209] Hydroxyethyl cellulose is a partially substituted poly(hydroxyethyl) ether of cellulose, a non-ionic hydrophilic polymer with linear and fibrous chains, having a degree of substitution of at least 1.5 (three substituted hydroxyl groups per two units)

[67] .

[0210] Triethanolamine is a tertiary amino compound, which is an amine in which each hydrogen is replaced by a 2-hydroxyethyl group. It has the function of a buffering agent and a 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.

[0211] PEG 400 is a low molecular weight grade of polyethylene glycol. It is a clear, colorless, viscous liquid. PEG 400 is widely used in various pharmaceutical formulations due in part to its low toxicity

[68] .

[0212] Methyl paraben is a 4-hydroxybenzoic acid ester, formed by the formal condensation of the carboxyl group of 4-hydroxybenzoic acid with methanol. It is the most commonly used antimicrobial preservative in cosmetics. In addition, it has the functions of a plant metabolite, an antibacterial food preservative, a neuroprotective agent, and an antifungal agent

[69] .

[0213] Evaluation of vesicle gel

[0214] The gel formulations were evaluated based on the following parameters:

[0215] Clarity: determined by visual inspection against a black and white background, graded as turbid, clear, and very clear (glassy).

[0216] Homogeneity: Determined by visually inspecting the appearance of the gel.

[0217] Sedimentation: Determined by visually inspecting for the presence of any aggregate.

[0218] In vitro drug release studies

[0219] In vitro dissolution studies of all prepared gels were performed using a Franz diffusion cell at 100 rpm and a temperature maintained at 37 ± 5 °C, with 50 mL of phosphate-buffered saline (pH 7.4) as the dissolution medium (Fathalla, 2014) (Helal et al., 2012). At specific time intervals over 48 hours, 0.5 mL aliquots were taken and the concentration of IRL-1620 was analyzed by HPLC. An equal volume of fresh medium was used after each sampling to maintain a constant volume. The samples were then mixed with 0.5 mL of acetonitrile:water (40:60, v / v) and analyzed by HPLC.

[0220] Stability Study

[0221] The stability of vesicle gel formulations was evaluated by storing them in sealed glass vials under two different storage conditions (i.e., refrigerated temperatures (2–8 °C) and room temperature) for 45 days (antas et al., 2016). After a period of study, samples were removed at different time intervals, and the percentage of gels was determined by HPLC.

[0222] result

[0223] Analysis of IRL-1620 (sovatide)

[0224] The developed high-performance liquid chromatography (HPLC) method for quantifying IRL-1620 is simple and sensitive. Separation is performed on a reversed-phase C18 column (250 mm × 4.6 mm), and the column elution is monitored at 215 nm using a UV detector. During the studies, the method was found to be precise, accurate, and specific. This method has been successfully applied to studies of encapsulation efficiency, drug release, and stability.

[0225] Characterization of vesicle complex

[0226] At 10x10 magnification, the microscopic view of the vesicle complex shows the presence of elliptical to spherical vesicles.

[0227] Encapsulation efficiency (EE)

[0228] Based on drug encapsulation studies, the maximum drug encapsulation efficiency was shown using F3. The encapsulation efficiencies for all formulations are as follows: Figure 17 What is represented.

[0229] The prepared vesicles exhibited high encapsulation efficiency. Formulation F3 showed the highest encapsulation efficiency of 93% for IRL-1620, indicating the optimal lipid quality required for vesicle formation. With further increases in lipid concentration, the encapsulation efficiency did not change significantly, suggesting that lipid concentration does not contribute to drug encapsulation into the matrix.

[0230] Table 5 Encapsulation efficiency of different formulations

[0231] Formulation Area % Encapsulation F1 77023 6.99 F2 612344 55.60 F3 1024288 93.0 F4 1022456 92.8 F5 1013652 92.04

[0232] Compatibility study

[0233] like Figure 18-20 As shown, the peaks in the IR spectrum of the drug-lipid mixture did not appear or disappeared, which confirms that there is no chemical interaction between the drug and the lipid.

[0234] Particle size analysis

[0235] Particle size analysis results showed that the prepared vesicles had a particle size in the range of 218 nm. Figure 21 Furthermore, increasing the proportion of soy lecithin does not change the size of the vesicles.

[0236] ζ-potential analysis

[0237] The zeta potential of the vesicles loaded with IRL-1620 is -35.1 mV, which is a measurement of the net charge of the vesicles. Figure 22 The negative charge on the surface of vesicles causes mutual repulsion between particles, stabilizing them and preventing aggregation (Bayindir & Yuksel, 2010). Therefore, vesicles have acceptable stability.

[0238] Evaluation of vesicle gel

[0239] Table 6 shows the appearance, color, homogeneity, and sedimentation results. G-3 was considered to have the best combination in terms of texture, homogeneity, and spreadability.

[0240] Table 6 Physical properties of vesicle gels

[0241] Formulation Appearance Color Homogeneity Precipitation G-1 Hard gel Clear Homogeneous None G-2 Gel Clear Homogeneous None G-3 Gel Clear Homogeneous None G-4 Gel Turbid Homogeneous None G-5 Viscous liquid Turbid Homogeneous None

[0242] In vitro drug release studies

[0243] In vitro studies of vesicle gel formulations were conducted using a Franz diffusion cell in PBS (pH 7.4) via goat skin (Shahiwala & Misra, 2002), and the results are shown below. Figure 24From the data, it was observed that there was a sharp increase in the in vitro release of IRL-1620 in the niosomal gel formulation within 6 hours; followed by a steady concentration and gradual decrease in release up to about 26 hours. The cumulative release of IRL-1620 from the formulation was 81.1% after 24 hours Figure 25 ).

[0244] Stability study

[0245] A stability study was performed by storing the niosomal gel formulation under different storage conditions for one month. At different time points, samples were withdrawn and IRL-1620 was determined using the HPLC method, the results of which are shown in Figure 26 .

[0246] From the results, it was observed that there was a decrease in drug content at room temperature; whereas, no significant change was observed at refrigerated temperature. Thus, at the end of the study, it was found that the developed niosomal formulation was stable under storage conditions.

[0247] Conclusion

[0248] The niosomal encapsulation efficiency of IRL-1620 was 6.99% to 93.0%. The niosomal formulation showed a particle size in the range of 218 nm. The compatibility study, zeta potential, and release study data indicated that by encapsulating the drug into niosomes, the release of the drug could be maintained and controlled for a longer duration of time, maintaining high stability. The use of hydroxyethyl cellulose and triethanolamine as a gel base allowed for better dispersion and release of IRL-1620 in the niosomes. Thus, the developed formulation had the ability to encapsulate IRL-1620, release the drug for a longer duration of time, and enhance the stability of the peptide moiety.

[0249] Test

[0250] In any of the above embodiments, testing is required to quantify the regeneration of damaged tissue. Visual granulation and epithelialization of the wound bed can be quantified strategically to assess wound healing. Microscopes, such as electron microscopes, can measure in vitro samples of tissue, thereby quantifying neuroepithelial and epidermal structures. If quantification of sensory function loss is required, a two-point discrimination test can be employed. Thermal stimulation of the skin can also be used to test for nerve recovery. Another test that directly measures nerve conduction, such as electromyography or electroneurography, can be used.

[0251] A comparative study was performed with two preferred embodiments regarding acute wound healing.

[0252] Title: Pharmacological evaluation of topical gel and biodegradable hydrogel film for local wound healing activity in excisional and incisional models of wounds in rats

[0253] Study details: This study was conducted to investigate the effect of treatment on wound healing in rats. The study involved excisional and incisional models of wound healing.

[0254] List of abbreviations: IHC: Immunohistochemistry; MT: Masson's trichome; TEM: Transmission electron microscopy; bFGF: Basic fibroblast growth factor; PDGF: Platelet-derived growth factor.

[0255] GLP Compliance Statement: This study was conducted following the principles of the current Good Laboratory Practice.

[0256] Excision model

[0257] Objective:

[0258] Induction of excisional wounds and evaluation of wound healing activity of test formulation as compared to standard pharmaceutical formulation based on the following parameters o Wound area

[0259] o Time of epithelialization

[0260] o Wound contraction rate

[0261] o Wet granulation tissue weight

[0262] o H&E, MT staining and IHC

[0263] Materials & Methods:

[0264] Animals: The protocol of the study was reviewed and approved by the Institutional Animal Ethics Committee of Delhi Pharmaceutical Sciences and Research University (DPSRU), New Delhi, following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Delhi, India. Albino Wistar rats weighing 180-200 g were used in the study. The animals were acclimatized for one week in the institutional animal house before use. The animals had free access to food and water ad libitum.

[0265] Study Groups:

[0266]

[0267]

[0268] The animals were acclimatized for 7 days prior to the study.

[0269] The Wistar albino rats were anesthetized with ketamine hydrochloride (50 mg / kg, i.p.), their dorsal skin was shaved using a depilatory cream and cleaned with 70% ethanol; a standard ring was used to mark an area of approximately 500 mm 2 of the rat's back, which was then carefully cut through the entire thickness using a surgical knife.

[0270] • In placebo 1 & treatment 1 groups, placebo 1 and gel were applied topically to rats for 16 days, respectively. In placebo 2 & treatment 2 groups, placebo 2 and biodegradable hydrogel film were applied every third day, respectively, covered with cotton and then gauze. In positive control group, animals were applied with standard drug.

[0271] • The wound induction day was considered as day 0.

[0272] • The wound area was measured on days 4, 8, 12 and 16 and the wound contraction rate was determined.

[0273] • On day 17 of the study, animals were sacrificed and tissues were collected for H&E, IHC and MT staining.

[0274] Observations:

[0275] On day 5, no epithelialization of the skin was observed.

[0276] Wound size was observed on days 4, 8, 12 and 16 (Table 7).

[0277] The day when eschar shedding without leaving a sore mouth was observed (Table 8)

[0278] The following parameters were observed:

[0279]

[0280]

[0281] All data are expressed as mean ± SD compared to disease control, *p < 0.05, **p < 0.01, ***p < 0.001; compared to placebo 1, # p < 0.05, ### p < 0.001; compared to placebo 2, @ p < 0.05, @@@ p < 0.001

[0282] Statistical analysis report: Overall, treatment groups had improved wound contraction rate compared to their respective placebo groups. Positive control significantly improved wound contraction rate compared to disease control. However, treatment 2 (film) did not find improvement in wound contraction rate compared to placebo 2 (film), but when combined with standard drug, it had a significant impact on wound contraction rate.

[0283] 2) Epithelialization phase

[0284]

[0285] All data are expressed as mean ± SD, *p < 0.05 compared to disease control; p < 0.05 compared to placebo 1, # p < 0.05 compared to placebo 2, @ p < 0.05

[0286] Statistical analysis report: Standard drug has significantly improved re-epithelialization rate compared to disease control. Treatment significantly improved re-epithelialization rate compared to respective placebo. However, treatment 2 (membrane) did not find improvement in re-epithelialization period compared to placebo 2 (membrane); however, it significantly affected re-epithelialization period when combined with standard drug.

[0287] 3) Wet granulation tissue weight

[0288] Veterinary opinion was that collection of granulation tissue would hamper H&E results; therefore, wet granulation tissue weight was not evaluated in this study.

[0289] In Figure 27: "II" represents inflammatory cell infiltration; "B" represents blood vessels; "An" represents angiogenesis; "FP + " represents fibroblast proliferation; "E" represents epidermis; "H" represents histiocytes. "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0290] Histological evaluation

[0291] The skin was evaluated histologically by H&E staining. Neutrophil infiltration and dense inflammatory cell infiltration (II) were observed in placebo 1, placebo 2, and disease control groups. However, moderate inflammation was observed in treatment groups. Angiogenesis, fibroblast proliferation, and collagen re-epithelialization were not observed in placebo 1, placebo 2, and disease control groups. Different treatments improved angiogenesis, fibroblast proliferation, and collagen deposition (Figure 27).

[0292] In Figure 28, "C" represents collagen; "FC" represents fibrous connective tissue. "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0293] MT staining: In disease control, placebo 1, and placebo 2 groups, a mild increase in collagen deposition was observed, while in respective treatment groups, an improvement in collagen deposition was found with treatment. In positive control, treatment 1+std, and treatment 2+std groups, an increase in collagen deposition throughout the dermis was found (Figure 28).

[0294] In Figure 29: "A, B, C, D, E, F, G and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std and treatment 2+std respectively.

[0295] In Figure 30: "A, B, C, D, E, F, G and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std and treatment 2+std respectively.

[0296] Immunohistochemistry: In placebo 1, it was recorded that VEGF was negatively expressed in the fibrotic area throughout the dermis. However, in placebo 2 and disease control, weak cytoplasmic positivity of VEGF was observed in vascular endothelial cells (and some spindle cells). In treatment groups, higher expression levels of VEGF were observed. Further, in placebo 1, placebo 2 and disease control groups, weak expression of vWF was observed in vascular endothelial cells in the fibrotic area throughout the dermis (Figure 29). All treatments had improved expression of vWF; however, it was recorded that in treatment 2, treatment 1+std and treatment 2+std, strong cytoplasmic positivity of vWF in vascular endothelial cells (Figure 30).

[0297] Conclusion: Different treatments improved the wound healing process by improving inflammatory parameters, fibroblast proliferation and angiogenesis. Treatments had improved wound contraction rate and re-epithelialization rate. Compared to placebo 1, placebo 2 and disease control groups, treatments accelerated the wound healing process by improving collagen deposition and increasing expression of VEGF and vWF in the respective groups.

[0298] Incision model

[0299] Objective:

[0300] • Induction of incisional wounds and application of test drugs

[0301] • Measurement of wound breaking strength (WBS)

[0302] • Collection of animal tissues for H&E, IHC and MT staining.

[0303] Materials and Methods:

[0304] Animals: The experimental protocol of the present study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Delhi Pharmaceutical Sciences and Research University (DPSRU), New Delhi, following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Delhi, India. Albino Wistar rats weighing 180-200 g were used in the present study. Prior to use, animals were acclimatized for one week in the institutional animal house, where they had free access to food and water ad libitum.

[0305] Study groups:

[0306] Research group Treatment n 1 Disease control 6 2 Placebo-1 control (placebo gel) 3 3 Placebo-2 control (placebo film) 3 4 Positive control (Regen-D150) 6 5 Topical gel 6 6 Biodegradable hydrogel film 6 7 Topical gel + standard drug (Regen-D150) 6 8 Biodegradable hydrogel film + standard drug (Regen-D150) 6 Total 42

[0307] Method:

[0308] • The animals were acclimated to the environment for 7 days prior to the study.

[0309] • Wistar albino rats were anesthetized with ketamine hydrochloride (50 mg / kg, intraperitoneal injection), their backs were shaved using an electric shaver (10 watts) and cleaned with 70% ethanol.

[0310] • Two parallel 6 cm paraspinal incisions were made in the entire skin thickness, 1 cm lateral to the spinal midline.

[0311] • In the placebo 2, treatment 2 and treatment 2+std groups, the rats were applied with placebo film, biodegradable film and biodegradable film+Regen D, respectively.

[0312] • The wounds were sutured with interrupted sutures at 1 cm intervals and with surgical sutures. On the 7th day after injury, the sutures were removed.

[0313] • In the placebo 1, positive control, treatment 1 and treatment 1+std groups, the rats were applied with placebo gel, standard drug Regen-D, test gel and test gel+Regen-D, respectively, for 16 days.

[0314] • The wound induction day was considered as day 0.

[0315] • On the 17th day of the study, the animals were sacrificed and the tissues were collected for H&E, IHC and MT staining.

[0316] Observations:

[0317] • On day 7, a large amount of pus formation was observed in animals numbered 1, 2, 3 of placebo 1 and treatment 2, respectively.

[0318] • On day 7, a large amount of pus formation was observed in animals numbered 1, 2, 3 of treatment 2+std.

[0319] • On day 10, a large amount of pus formation was observed in animal numbered 3 of treatment 2.

[0320] • The wound breaking strength was recorded on the 10th day of the study (Table 9).

[0321] The following parameters were observed:

[0322]

[0323] All data are expressed as mean ± SD, ***p < 0.001 compared to disease control; ****p < 0.0001 compared to placebo 1, # p < 0.05 compared to placebo 2, @ p < 0.05, @@@ p < 0.05

[0324] Statistical analysis report: Both treatments improved the wound breaking strength compared to the corresponding placebo. However, the standard group had improved wound breaking strength compared to the disease control.

[0325] Histological evaluation (H&E)

[0326] In Figure 32: "II" represents inflammatory cell infiltration; "B" represents blood vessels; "An" represents angiogenesis; "FP + " represents fibroblast proliferation; "E" represents epidermis; "H" represents histiocytes. "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0327] Histological evaluation: Histological evaluation was performed by H&E staining. The placebo 1 group, placebo 2 group, and disease control group showed an increase in inflammatory parameters, whereas the different treatments reduced inflammation compared to the placebo 1 group and placebo 2 group. However, mild inflammation was observed in the treatment 1 group. In the treatment groups, angiogenesis and fibroblast proliferation increased. In addition, normal epidermis was observed in the treatment 1+std group and treatment 2+std group (Figure 32).

[0328] In Figure 33: "C" represents collagen; "FC" represents fibrous connective tissue. "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0329] MT staining: Slight increase in collagen deposition was observed in the placebo 1 group, placebo 2 group, and disease control group. Collagen deposition was slightly higher in the other groups compared to the placebo 1 group, placebo 2 group, and disease control group (Figure 33).

[0330] In Figure 34: "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0331] Immunohistochemistry: Determination of vWF expression

[0332] In Figure 35, "A, B, C, D, E, F, G, and H" represent disease control, placebo 1, placebo 2, positive control, treatment 1, treatment 2, treatment 1+std, and treatment 2+std, respectively.

[0333] Immunohistochemistry: Weak cytoplasmic positive expression of VEGF in vascular endothelial cells was observed in placebo 1, placebo 2, positive control, treatment 1, and treatment 2+std groups, while no VEGF expression in vascular endothelial cells was found in placebo 2 and disease control groups. Increased VEGF expression was recorded in treatment 2 and treatment 1+std groups. Figure 8 No vWF expression was found in placebo 2 and disease control groups. Weak cytoplasmic positive of vWF in vascular endothelial cells was recorded in placebo 1, positive control, treatment 1, treatment 2, and treatment 1+std groups, while higher vWF expression was observed in treatment 1+std group compared to other groups (Figure 35).

[0334] Conclusion: Treatments improved fibroblast proliferation, collagen deposition, and angiogenesis. Treatments had slightly improved VEGF and vWF levels in the respective groups. Treatments had significantly improved different parameters compared to placebo groups. In addition, the standard group had a significantly increased WBS compared to the normal control.

Claims

1. A method for treating dermal wounds or skin wounds and damaged cranial or peripheral nerves, said method using a compound containing an endothelin analogue to limit cell death and promote the formation of new neurons (neurogenesis) and new blood vessels (angiogenesis) through mechanisms of anti-apoptotic, anti-inflammatory and antioxidant activity, thereby enhancing wound healing and achieving nerve regeneration.

2. The method according to claim 1, wherein the endothelin B receptor analogue is selected from N-succinyl-[Glu] 9 Ala 11 ,15 Endothelin 1 (Sovastatin, IRL-1620), BQ-3020, [Ala 1,3,11,15 - One or more of endothelin, keratoxin S6c, and endothelin 3.

3. A composition comprising: (a) Endothelin B (ET) B Receptor agonists; (b) a growth factor, or an osmotic agent, or a diuretic, or a steroid, or transplanted stem cells, or ferulic acid, or arctiin, or espin isotype 1 polypeptide, or a neurotrophic factor selected from one or more of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, and ciliary neurotrophic factor, or human insulin-like growth factor 1, or Netrin 1, or a mixture thereof; wherein the osmotic agent comprises glycerol, the diuretic comprises furosemide or ethacrynic acid, and Optional (c) excipients.

4. An article comprising: (a) A packaged composition comprising endothelin B (ET) B ) receptor agonists and steroids or their salts; (b) An insert, said insert being provided for simultaneous or sequential application of the ET B Instructions for use of receptor agonists and the steroids or their salts for the treatment of patients; and (c) Containers used for (a) and (b).

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

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

7. The method of claim 2, wherein the endothelin B receptor agonist analogue is formed alone as a formulation, or the endothelin B receptor analogue is coupled with nanoparticles including elusive nanoparticles or hydrogels to form a formulation, the formulation comprising gels, creams, solutions, lotions, ointments, adhesive patches, and sprays.

8. The method of claim 2, wherein the endothelin B receptor agonist analog is administered via a sustained-release drug delivery system, the sustained-release drug delivery system comprising... Hyaluronic acid gel, Seprapack TM The sustained-release drug delivery system includes thiol-modified hyaluronic acid, glutaraldehyde crosslinking of porcine collagen, gelatin, chitosan glycosylated derivatives, poloxamer 407, and chitosan glycerol phosphate hydrogel. The sustained-release drug delivery system also includes lipid core nanocapsules of poly-L-lysine (HBPL) nanoparticles.

9. The method of claim 1, wherein the endothelin analogue, particularly the endothelin B receptor agonist analogue, is used to treat wounds and / or promote wound healing, and for the regeneration of damaged nerves in mammals.

10. The method of claim 1, wherein the endothelin receptor agonist analogue administration may be combined with a growth factor, a penetrant, a diuretic, a steroid, transplanted stem cells, ferulic acid, arctiin, or espin isotype 1 polypeptide, or a neurotrophic factor selected from one or more of glial cell-derived neurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, and ciliary neurotrophic factor, or human insulin-like growth factor 1, or Netrin 1, or mixtures thereof; the penetrant comprising glycerol, and the diuretic comprising furosemide or ethacrynic acid.

11. The method of claim 1, wherein the dose of the endothelin receptor agonist analog ranges from 0.00001 to 1 mg, and may be administered once or multiple times over a day, several weeks, or several months.

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