Use of GHRP-6 for the treatment of senescence and healing of complex chronic refractory wounds

Abstract

The present invention describes for the first time a method for preventing and / or reversing the phenomenon of premature senescence of skin cells and complex chronic wounds, characterized in that an effective amount of the growth hormone secretagogue peptide GHRP6 is applied. Also object of the present invention is a method for treating cases of complex chronic wounds that have developed primary or secondary refractoriness caused by the development of a cellular senescence process, transforming them from refractory wounds to responsive wounds in the presence of proliferative agents with which they unexpectedly manage to reverse and restart the healing process. Complex chronic wounds include cases of diabetic foot ulcers, venous ulcers, pressure ulcers, radiation ulcers, and other types of chronic wounds that are characterized by refractoriness to all types of treatment. These cases currently have no effective treatments.

Description

[0001] DESCRIPTIVE MEMORANDUM

[0002] METHOD FOR THE TREATMENT OF SENESCENCE AND HEALING OF COMPLEX REFRACTORY CHRONIC WOUNDS

[0003] Field of technology

[0004] The present invention falls within the fields of human medicine and the pharmaceutical industry. In particular, it relates to the use of the growth hormone secretagogue peptide GHRP6, which, in combination with healing agents, allows for the resumption of the healing process and the complete closure of complex, refractory chronic wounds.

[0005] Prior art

[0006] Chronic wounds impose a high burden of morbidity, mortality, and impaired quality of life, not to mention that, for example, in the United States, conservative estimates of the annual cost of treating and managing chronic ulcers exceeds $31 billion (Nussbaum SR., et al. An economic evaluation of the impact, cost, and Medicare policy implications of chronic nonhealing wounds. Value in Health 2018; 21 (1): 27-32).

[0007] Complex wounds are defined as those involving full-thickness skin loss where the base of the injury may be muscle tissue, fascia, tendons, bone, or supporting structures, and which, due to their special characteristics, may compromise their normal healing process, despite appropriate treatment with conventional methods and, where possible, the elimination of the underlying cause (Ferreira MC., et al. Complex wounds. Clinics 2006;61 (6):571-578). Chronic wounds, which are generally complex due to the anatomy of tissue loss, are defined as those whose healing extends for 6 weeks or more, whose closure process is interrupted or arrested due to intrinsic and extrinsic factors, with heterogeneity and asynchrony in the closure phases (https: / / www.salusplaY.com / apuntes / heridas-cronicas--y--ulceras-por-presion / tema-1 - heridas-crónicas).

[0008] The main clinical forms of these chronic wounds are diabetic foot ulcers, pressure ulcers, and venous or varicose ulcers. These lesions can remain unhealed for years, and lower limb ulcers in the diabetic population are the leading cause of amputation (Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4-14). Interestingly, some of these lesions begin the healing process, which then suddenly stops without an apparent clinical cause, becoming refractory to any intervention. The underlying molecular pathological basis for the refractoriness, and therefore the chronicity, of these wounds is not fully characterized despite years of research and dedicated effort (Gould L, Abadir P, Brem H, et al. Chronic wound repair and healing in older adults: current status and future research).J Am Geriatr Soc 2015;63:427-438).

[0009] Diabetes mellitus (DM) is a global public health threat, having expanded dramatically in the last two decades. Epidemiological studies indicate that the number of patients with DM will reach more than 360 million people by 2030 (Yazdanpanah L, et al. World J Diabetes. 2015 Feb 15;6(1):37-53. doi: 10.4239 / wjd.v6.¡1 .37.). Patients with DM progress through a variety of multi-organ complications, in which diabetic foot ulcer (DFU) is one of the most important and most feared complications (Pastar, I et al. In: Veves, A., Giurini, J., Guzman, R. (eds) The Diabetic Foot. Contemporary Diabetes. Humana, Cham, https: / / doi.org / 10.1007 / 978-3-319-89869-8_7).

[0010] Diabetes affects all cellular and molecular processes involved in wound healing, leading to the development of a chronic wound phenotype. Since diabetes is a systemic and multi-organ disease, comorbidities act as limiting factors for healing and must be addressed by multidisciplinary teams to ensure timely healing and reduce amputation rates (Burgess, JL et al. Medicina 2021, 57, 1072. https: / / doi.org / 10.3390 / medicina57101072). The lifetime risk of developing a diabetic foot ulcer has been estimated to be between 19% and 34%. It is also estimated that between 9.1 and 26.1 million people with diabetes worldwide develop foot ulcers each year (Micheál Edmonds et al. Journal of Clinical Orthopaedics and Trauma Volume 17, June 2021, Pages 88-93).Diabetic foot ulcers (DFUs) are a debilitating and disabling complication that has steadily increased over the years. They are also the leading cause of non-traumatic lower limb amputations, with a diabetic patient undergoing amputation every 20 seconds. Five-year survival rates for amputee patients are lower than those of five types of fatal malignant diseases (Dane K. Wukich, et al. Foot & Ankle Specialist 2017;11:1, 17-21).

[0011] A latent challenge that is frequently encountered and has no remedy so far is the sudden and unexpected arrest of the healing process once it has begun, so that the lesion may become granulated but not re-epithelialized and, consequently, evolves into a state of "suspended chronicity" in which a recapitulation or re-emission of the chronic episode has occurred (Yazdanpanah L, Nasih M, Adarvishi S. Literature review on the management of diabetic foot ulcer. World J Diabetes. 2015 Feb 15;6(1):37-53. doi: 10.4239 / wjd.v6.i1.37. PMID: 25685277; PMCID: PMC4317316).

[0012] Diabetic foot ulcers (DFUs) are responsible for dramatic emotional and physical deterioration that limits quality of life, as well as substantial financial losses. Diabetic foot syndrome represents an enormous economic burden worldwide, accounting for between 7% and 20% of total diabetes expenditures in North America and Europe. The social impact of diabetic foot is estimated to exceed one billion dollars annually. Despite advances in sophisticated treatment modalities, ulcers that are resistant to healing from the outset or that become chronic by halting the healing process once it has begun remain a significant threat to lower limb preservation and patient survival. Therefore, current treatments remain suboptimal, and further research is needed.Pressure ulcers (PUs) are characterized by a complex and intricate molecular pathophysiology that appears to stem from poorly controlled blood glucose levels. Glucotoxic stress impairs the healing response and disrupts the flow of overlapping healing phases, ultimately promoting a "chronic wound phenotype" that is further exacerbated by the presence of a large population of senescent cells. Consequently, PUs are considered "recalcitrant chronic wounds" because they have failed to progress through the normal phases of healing and thus enter a state of pathological inflammation. As a result, the healing process is delayed, incomplete, and uncoordinated, leading to a poor anatomical and functional outcome (Ancha Kishore Babu, M. et al. (2022). International Journal of Health Sciences, 2835-2850).In the case of low-grade diabetic neuropathic ulcers, complete wound closure occurs after 9 months, representing an immeasurable loss of time and money. More complex wounds, such as those with an ischemic component, exhibit even longer healing periods under a modern standard care regimen (the average duration is 133 days), while some patients never achieve wound healing (Robson MC, et al. Arch Surg. 2000;135(7):773-777. doi:10.1001 / archsurg.135.7.773). A set of medical interventions aimed at reversing the chronic wound phenotype of UPD can trigger the resumption of the healing process and ultimately achieve epithelialization in 70% of cases for low-grade lesions (Spampinato, SF, et al. Pharmaceuticals 2020, 13, 60. https: / / doi.org / 10.3390 / ph13040060).However, a distinctive feature of pressure ulcers and other types of chronic wounds is the unpredictable arrest of the healing process once the wound has progressed to granulation tissue formation, contraction, and epithelial migration, thus triggering a new episode of chronicity. This phenomenon is known as secondary refractoriness. This state, in which the ongoing healing process inexplicably stops, is followed by therapeutic failure, as patients respond very little to treatment. Multiple molecular hypotheses have been put forward, including the recently coined term "cell jam," to describe the collective arrest of cell movement that gives rise to a characteristic epidermal rim at the base of the ulcer (Elizabeth Lawson-Keister, M. Lisa Manning. Current Opinion in Cell Biology. Volume 72, October 2021, Pages 146-155), (Aldana, PC, Khachemoune, A. Am J Clin Dermatol 21, 255-264 (2020). https: / / doi.(org / 10.1007 / s40257-019-00495-x). Clinical experience shows that these wounds, which recapitulate a state of "re-chronification," have a worse prognosis, a much more protracted evolution (which can last for years), require a greater number of invasive surgical debridements, and contribute to a higher number of amputations. Consequently, this dreaded event within the biology of chronic wounds, especially pressure ulcers, remains mechanistically unexplained and, therefore, lacks effective treatment (Monika P, et al. Human primary chronic wound derived fibroblasts demonstrate differential pattern in expression of fibroblast specific markers, cell cycle arrest and reduced proliferation. Experimental and Molecular Pathology Volume 127, August 2022, 104803). A unique feature of these chronic recurrent ulcers is that they act as an important portal of infection, which explains a high risk of amputations (Ramírez-Acuña, JM, et al.Antibiotics 2019, 8, 193. https: / / doi.org / 10.3390 / antibiotics8040193).

[0013] These lesions also exhibit a higher incidence of re-ulceration, which increases the amputation rate compared to pressure ulcers that do not stop healing (Rayman G, et al. Diabetes Metab Res Rev. 2020; 36(S1): e3283. https: / / doi.org / 10.1002 / dmrr.3283). These factors highlight the need for novel interventions and effective treatments for this unique wound subpopulation and for pressure ulcers in general (Huang Y, et al. JAMA Netw Open. 2021;4(9): e2122607. doi:10.1001 / jamanetworkopen.2021.22607).

[0014] Currently, there is a group of innovative approaches to enhance the healing of chronic diabetic wounds. All of these modern approaches build upon the traditional principles already described. Some of the new methods include:

[0015] • Tissue engineering and other forms of skin substitutes. These are allogeneic cellular or acellular dermal grafts. These products can consist of fibroblasts and keratinocytes cultured in collagen matrices, as a type of two-layered living skin construct. Despite advances in the treatment of chronic wounds with bioengineered skin, there are still almost 50% of patients who do not heal when their ulcers have previously been resistant to conventional therapy (Hadi Samadian, et al. (2018). Artificial Cells, Nanomedicine, and Biotechnology 46:sup1, pp. 964-974).

[0016] • Growth factors (GFs). Early attempts to administer GFs to chronic wounds were likely driven by the concept of "replacement therapy," aiming to restore the biological competence of local cells and ultimately resume the physiological healing pathway. Growth factors play a significant role in local inflammation, re-epithelialization, granulation, tissue formation, neovascularization, and the production of extracellular matrix from various cellular sources and through diverse mechanisms. The environment of pressure ulcers has been shown to significantly impair the response to GFs by hindering receptor binding and subsequent activation (Pastar I. (2010) Attenuation of the transforming growth factor beta-signaling pathway in chronic venous ulcers).Mol Med 16: 92-101), (Liu Y, Liu Y, Deng J, Li W, Nie X (2021) Fibroblast Growth Factor in Diabetic Foot Ulcer: Progress and Therapeutic Prospects. Front Endocrinol (Lausanne) 12: 744-868), (Ren X (2019). Front Bioeng Biotechnol 7: 469). Historically, it has been necessary to modify local wound factors to ensure an adequate pharmacodynamic response of GFs and prevent their degradation after topical application in diabetic foot ulcers and other chronic wounds (Sibbald RG, et al. (2021). Adv Skin Wound Care 34: 183-195), (Berlanga-Acosta J, et al. (2020) MEDICC Rev 22: 24-31). To date, treatment with GFs has not met initial expectations.

[0017] • Gene therapy. This technique involves introducing the gene instead of the product (growth factor), which is considered cheaper and more effective for treating non-healing wounds. More studies are needed to translate preclinical enthusiasm into clinical results (Davis, FM, et al. Curr Diab Rep 18, 2 (2018). https: / / doi.org / 10.1007 / s11892-018-0970-z).

[0018] • Stem cell therapy. A recently published meta-analysis indicates that stem cell-based therapy can improve the healing of diabetic foot ulcers and is associated with less pain, a lower amputation rate, and a better prognosis compared to standard treatment. However, further studies are needed to fully consider this approach as a novel therapy (Xuan Shu, et al. Endocrine Journal. Vol. 65 (2018), No. 4 pp. 403-413. https: / / doi.org / 10.1507 / endocrj.EJ17-0424).

[0019] • Occlusive dressing technology. These are designed to preserve wound moisture, which has been shown to improve the healing process by stimulating the migration and proliferation phases that lead to increased epithelialization and angiogenesis (Schaper, NC, et al. Diabetes Metab Res Rev. 2020; 36 (S1):e3266. https: / / doi.org / 10.1002 / dmrr.3266). However, this technology is very ineffective in complex, refractory wounds.

[0020] • Hyperbaric oxygen therapy (HBOT). Although HBOT is widely used in the treatment of diabetic foot ulcers (DFUs), there is still much controversy regarding its efficacy in DFUs (Lóndahl, M, Boulton, AJM. Diabetes Metab Res Rev. 2020; 36(S1):e3233. https: / / doi.org / 10.1002 / dmrr.3233). While the International Working Group on Guidelines for Diabetic Foot Wound Healing suggested considering HBOT for non-healing DFUs, the strength of this recommendation is weak, and many cases do not achieve adequate closure (Rayman G, et al. Diab Metab Res Rev. 2020;36(S1):e3283).

[0021] • Negative pressure wound therapy (NPWT). Some clinical trials have suggested that NPWT is an effective and safe method for promoting healing of diabetic foot ulcers. However, serious complications related to this treatment have been reported in recent years. It also appears to be more expensive than conventional methods for treating diabetic foot ulcers (Liu S, et al. Ther Clin Risk Manag. 2017 Apr 18; 13:533-544. doi: 10.2147 / TCRM.S131193).

[0022] Historically, there has been a lack of evidence supporting topical therapy for accelerating the healing of diabetic foot ulcers (DFUs), and in this regard, the use of growth factors remains controversial (Dixon, D., Edmonds, M. Managing Diabetic Foot Ulcers: Drugs 81, 29-56 (2021). https: / / doi.org / 10.1007 / s40265-020-01415-8). As mentioned previously, there is compelling evidence that topically administered growth factors degrade in situ, limiting their clinical efficacy. To avoid local degradation of epidermal growth factor (EGF), ensure its local diffusion, and deliver it to the responding cell populations in depth, the intralesional EGF infiltration method was developed. This technique has led to a reduction in lower limb amputations in diabetic patients (WO 03 / 053458).The intervention procedure is based on infiltration into the wound bed and surrounding areas of complex, high-grade, neuropathic, and ischemic diabetic wounds of the lower extremities, including both ulcers and stagnant amputation residual sites (Berlanga-Acosta J, Rodríguez HC, Mari YM, Cama VF, Ojalvo AG, et al. (2020). MEDICC Rev 22: 24-31). However, this infiltration therapy has demonstrated limited utility in reversing diabetic wounds that re-occur after initial healing. Regardless of the efficacy of this therapy and treatment method, particularly in filling the wound area with productive granulation tissue, experts in the field emphasize the need for an additional complementary approach to stimulate wound closure through epithelialization.

[0023] Despite the progress shown with EGF infiltration therapy and other current therapies, the complete healing and closure of complex ulcers refractory to all treatments remains unresolved, with serious consequences that can lead to amputation.

[0024] A new class of agents for the protection and repair of cells and tissues, known as growth hormone secretagogues (GHS), has recently emerged. GHS are a variety of synthetic peptide and non-peptide agents that stimulate the endogenous release of growth hormone (GH). Treatment with GHS increases serum levels of GH and insulin-like growth factor 1 (IGF-1) (Sinha DK, et al. Transí Androl Urol. 2020 Mar; 9 (Suppl 2):S149-S159. doi: 10.21037 / tau.2019.11.30. PMID: 32257855; PMCID: PMC7108996). Growth Hormone-Releasing Peptide-6 (GHRP-6) is a peptide member of the GHS family and a promising molecule (H-His1-d-Trp-Ala-Trp-d-Phe-Lys6-NH2) for the treatment of various wasting and debilitating diseases, including heart failure, stroke, sarcopenia, etc. (Berlanga-Acosta J. Cardiology Volume 11: 1-9. 2017. DOI: 10.1177 / 1179546817694558).

[0025] The ability of the growth hormone secretagogue peptide GHRP-6, when co-administered with epidermal growth factor, to act as a rescue agent for organs and tissues subjected to total or partial ischemia in internal viscera has been reported (Pharmaceutical combination characterized in that it comprises the administration of a peptide with epidermal growth factor-like activity together with the growth hormone secretagogue peptide GHRP-6; US Patent US7361638B2). However, the mechanisms related to tissue damage due to lack of arterial blood supply are not related in their pathophysiology, natural course, and prognosis to the refractory nature of wound healing and the chronicity of wounds in peripheral tissues.Hence, its effect and application has been directed only to systemic use in intensive care units in critically ill patients with a high risk of death due to multiple organ dysfunction, respiratory distress syndrome, hypoxic neonates, hypovolemic shock and necrotizing enterocolitis, primarily.

[0026] Unlike acute wounds, the microenvironment of chronic human wounds is hostile to the physicochemical stability of hormones, receptors, and growth factors. It is characterized by a pleomorphic phenotype and marked asynchrony in the succession of stages in the healing process. In principle, unlike acute lesions, ulcers are long-standing, generally frozen in the inflammatory stage, with plasmarrhea or exudation of high degradative or proteolytic capacity, highly pro-oxidant, torpidly granulated, and poorly contracted (P. Martin, R. Nunan, Cellular and molecular mechanisms of repair in acute and chronic wound healing, British Journal of Dermatology, Volume 173, Issue 2, 1 August 2015, Pages 370-378, https: / / doi.org / 10.1111 / bjd.13954).In many cases, these lesions are poorly perfused with arterial blood, creating a microenvironment of hypoxic cells that initiate a program of arrest and premature senescence. This ultimately leads to the formation of a community of prematurely aged cells that promotes the creation of a polymicrobial biofilm, further contributing to the arrest of wound healing (Nunan, R., Harding, KG, & Martin, P. (2014). Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity. Disease models & mechanisms, 7(11), 1205-1213). All of these characteristics are endogenous factors inherent to the aging of the organism, or are consequences of chronic and debilitating diseases such as diabetes mellitus or sarcopenia.

[0027] There is evidence that cellular senescence plays a significant role in the pathophysiology of chronic wounds and that preventing or eliminating cellular senescence can restore the healing process, avoiding refractoriness and chronicity. It is known that senescent cell communities reside within chronic wounds, leading to persistently elevated secretion of inflammatory mediators and free radicals, and arresting cell proliferation. All of this affects granulation, angiogenesis, matrix remodeling, plasticity, and cell regeneration (Xuerong Wei, Minxiong Li, Zijun Zheng, Jun Ma, Yanbin Gao, Lianglong Chen, Yujie Peng, Shengxiang Yu, Lei Yang, Senescence in chronic wounds and potential targeted therapies, Burns & Trauma, Volume 10, 2022, tkab045, https: / / doi.org / 10.1093 / burnst / tkab045).

[0028] Cellular senescence is a response to multiple types of cellular stress, including DNA damage, telomere erosion, oncogene activation, oxidative damage, protein misfolding, and exposure to extracellular signals (such as mitogens and cytokines). At the molecular level, p53-Ser 15, p16INK4a / Rb, and p21 CIP1 are central to the regulatory pathways of senescence and cell arrest (Thompson, Elizabeth L. Ph.D.; Pitcher, Louise EBS; Niedernhofer, Laura J. Ph.D., MD; Robbins, Paul D. Ph.D. Targeting Cellular Senescence with Senotherapeutics: Development of New Approaches for Skin Care. Plastic and Reconstructive Surgery 150: 12S-19S, October 2022. doi: 10.1097 / PRS.0000000000009668).

[0029] The hypothesis that cellular senescence is an imperceptible underlying force in the pathogenesis of refractoriness and chronicity of diabetic wounds, as well as their recurrence, has gained traction in recent years (Berlanga-Acosta J, et al. Front. Endocrinol., 16 September 2020 Sec. Clinical Diabetes Volume 11 - 2020 https: / / doi.org / 10.3389 / fendo.2020.573032). Therefore, the failure of wound healing associated with diabetes and other complex ulcers, and the reduction in the resilience of scar tissue, are clinical manifestations of the negative impact of a community of senescent cells entrenched in the lesion, capable of self-perpetuation and propagation. Diabetes mellitus, with its associated hyperglycemia, is a pro-senescence disease.Hyperglycemia, oxidative stress, mitochondrial and DNA damage, as well as the excessive formation and accumulation of advanced glycation end products, are the main triggers for the senescent phenotype. Consequently, diabetic foot ulcers are definitively affected by a society of aged cells, rich in dormant or "stunted" fibroblasts, which expands and perpetuates itself, leading to refractoriness and perpetuating the chronic phenotype. This society is amplified by an archetypal secretome that induces replicative senescence in dermal fibroblasts, endothelial cells, and keratinocytes. Mesenchymal stem cells are also susceptible to the main drivers of diabetic senescence, which explains the inability of these cells to contribute to healing (Berlanga-Acosta J, et al. Front. Endocrinol., 16 September 2020 Sec. Clinical Diabetes Volume 11 - 2020 | https: / / doi.org / 10.3389 / fendo.2020).573032).

[0030] Recently, it has been discovered that other forms of complex chronic wounds, such as venous, radiation, and pressure ulcers, also exhibit a deeply rooted community of senescent cells that impose a pro-oxidative, degradative, inflammatory, and apoptogenic phenotype that blocks the healing mechanism and may underpin refractoriness. Therefore, cellular senescence appears to be an important pillar for the chronicity of wounds (Berlanga-Acosta J, et al. Front. Endocrinol., 16 September 2020 Sec. Clinical Diabetes Volume 11 - 2020 | https: / / doi.org / 10.3389 / fendo.2020.573032).

[0031] However, to date, no effective way has been found to eliminate cellular senescence in complex chronic wounds and achieve an effective treatment to break the refractoriness that this condition entails in the healing and closure of the wounds.

[0032] The full-thickness wound in rats is a recommended and appropriate model for evaluating the rate of closure and each of the biological events integrated into the healing process, such as granulation, angiogenesis, epithelialization, contraction, and remodeling, which can be modified by the external administration of a substance (Dorsett-Martin, WA (2004). Rat models of skin wound healing: a review. Wound repair and regeneration, 12(6), 591-599). However, in this model, the topical administration of GHRP-6 has not been shown to stimulate wound closure in normal rats with acute, clean, full-thickness wounds. Nevertheless, a novel finding in terms of the biological properties of this agent, reported in this invention, is its ability to reverse or dismantle the senescent phenotype in cultured fibroblasts derived from skin ulcers of different classifications.Thus, GHRP-6 may be a cooperating agent in healing by reducing the burden of senile fibroblast populations, which can subsequently respond to healing agents.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention solves the problem posed above based on unpublished results obtained regarding the effects of exposure of complex chronic wounds to growth hormone-releasing peptide (GHRP-6).

[0035] The present invention describes the effects of the peptide GHRP-6 on suppressing or reversing the molecular effectors of cellular senescence in cutaneous fibroblasts ex vivo and in vitro, transforming them from refractory to responsive to proliferative agents such as hormones and growth factors or other healing therapies. In other words, GHRP-6 reverses cell arrest and enables subsequent proliferation in response to a second agent—such as a mitogen.

[0036] In this scenario, GHRP-6 attenuated nuclear translocation and p53-Ser 15 expression, restored the expression of Cyclin D1, PGC-1, and SIRT1, preventing or reversing cell arrest, wound chronicity, and refractoriness by blocking senescence signaling pathways. These results are even more unexpected given that the state of the art demonstrates that the environment of the complex chronic wounds referred to in the present invention impairs the response to peptide molecules by preventing the binding and subsequent activation of their receptors due to the presence of local wound factors that do not guarantee an adequate pharmacodynamic response and promote their degradation after topical application.

[0037] The present invention relates to a method for treating cases that have developed primary or secondary refractoriness, with which they unexpectedly manage to reverse and restart the healing process of complex chronic wounds that have arrested and recapitulate a new event of chronicity when they were already progressing in their healing after weeks with treatments described in the state of the art.

[0038] The method described in the present invention succeeds in reversing secondary refractoriness in complex chronic wounds, allowing the healing process to be completed, even surprisingly, with the same healing treatment used prior to the sudden and unexpected stoppage of the healing process when it had already begun.

[0039] In a new embodiment of the present invention, a treatment method is described for initiating the healing of complex chronic wounds with primary refractoriness to treatments established in the state of the art.

[0040] In a preferred embodiment, the present invention describes a pharmacological treatment method based on two independent ingredients, targeting a particular subpopulation of chronic ulcers that, having initiated the healing process, unexpectedly fail to maintain the contraction and epithelialization processes, arresting the closure pathway and leading to a second episode of chronicity. This subtype of wound comprises the presence of aged and thickened collagen fibers, with abundant senescent fibroblasts. The treatment method is based on the simultaneous co-administration of the secretagogue agent GHRP-6 to the wound bed and contours, along with the administration of epidermal growth factor infiltrated into the ulcer or another healing agent.This co-administration is justified by the understanding that neither ingredient alone is capable of restoring the wound's contraction and epithelialization processes. Although this subgroup of wounds is inherently much more resistant to contemporary treatments, the combination of topical GHRP-6 and infiltrated EGF reactivated and sustained the healing mechanisms until the wound closed. The first ingredient, as already mentioned, reversed the senile phenotype, making the wounds responsive to the second—which, due to its mitogenic nature, does stimulate healing.

[0041] The present invention describes the use of the growth hormone secretagogue peptide GHRP6 to eliminate primary or secondary refractoriness and restart the healing process of complex chronic wounds that have arrested and recapitulate a new chronic event after having progressed in healing following weeks of highly effective treatments reported for these cases in the prior art. In specific embodiments, the present invention describes the combined use of the growth hormone secretagogue peptide GHRP6 with wound healing therapies to eliminate primary and secondary refractoriness of complex chronic wounds and simultaneously allow the restart of the healing process.

[0042] The complex chronic wounds of the present invention include, among others, diabetic foot ulcers, venous or varicose ulcers, pressure ulcers, or radiation ulcers.

[0043] In new examples of specific embodiments, the treatment method of the present invention combines treatment with the secretagogue peptide to reverse cell arrest and chronicity of wounds with hyperbaric oxygen treatments and patients treated with a vacuum-assisted closure machine when the latter, having initiated the healing process, unexpectedly fail to maintain the contraction and epithelialization processes, arresting the closure trajectory and recapitulating a second episode of chronicity.

[0044] Examples of implementation

[0045] Example 1. Infiltrated EGF and topical application of GHRP-6 reverse the recurrence of chronicity or secondary refractoriness and promote wound closure.

[0046] The study was conducted in a cohort of 12 diabetic patients admitted to the diabetic angiopathy ward of the National Institute of Angiology and Vascular Surgery, where the infiltrated EGF protocol is routinely applied unless contraindicated by specialists. These patients were included in this study because they experienced a sudden arrest of the healing process after all their lesions had begun to evolve satisfactorily and stably. Initially, the patients presented with high-grade lower extremity wounds, including ulcers and residual amputation bases (RABs) of neuropathic and ischemic origin, grades 3 and 4 on the Wagner scale. The RABs corresponded to patients who had undergone previous surgical procedures, including transmetatarsal amputations, toe disarticulations, and drainage of plantar abscesses with removal of local necrotic tissue.All patients were under treatment to control blood glucose levels and received systemic antimicrobial therapy. Other palliative interventions were also implemented to manage hydration levels, blood pressure, and provide analgesics. Wounds were debrided, cleaned, and bandaged with saline solution. Dressings and other subsequent local procedures were performed every two days. Once the wounds were clinically free of infection and the patients were metabolically stable, they were enrolled in the recombinant human EGF infiltration protocol, administered three times per week to accelerate wound closure. Consequently, each wound received 75 pg of EGF per treatment session (Fernandez-Montequin J. et al. Int Wound J. 2009 Dec;6(6):432-43. doi: 10.1111 / j.1742-481X.2009.00641.x).

[0047] Three weeks after initiating EGF treatment, corresponding to nine infiltration sessions, both the ulcers and the neuropathic and ischemic recurrent aortic ruptures (RABs) showed varying percentages of coverage with productive granulation tissue, encompassing approximately 40% to 65% of the affected area. Treatments and wounds progressed satisfactorily until weeks 5 to 7, when some showed clinical signs of atonicity and wound edge arrest. This resulted in the cessation of granulation tissue growth, contraction, and epithelial migration. These wounds were not infected, and analysis of the patients' medical records provided no evidence to elucidate the underlying cause of the halted healing. Table 1 reflects the main characteristics of the patients' wounds, as well as the number of EGF infiltration treatments until the lesions reached a state of wound arrest.Table 1. Characteristics of wounds and treatments with EGF up to the time of the scar arrest state.

[0048] (*) Represents the number of EGF infiltration sessions received up to the point of cessation of progression. Once wound arrest was confirmed, patients continued to receive EGF infiltrations every other day as routinely done, along with other standard medications. At this point, topical co-administration of GHRP-6 at a dose of 400 pg / ml in physiological saline solution was added. This dose was derived from experiments that demonstrated its cardioprotective (Berlanga-Acosta J et al. Clinical Science (2007) 112, 241-250), systemic cytoprotective (Cibrian-Vera D. Applied Biotechnology 2008; Vol. 25, No. 3), and antifibrotic effects in different animal models (Berlanga-Acosta J, et al. Applied Biotechnology 2012; 29: 60-72).

[0049] Nine to twelve days after initiating this co-administration, all wounds had resumed the healing process, exhibiting reddish granulation tissue growth and a progressive reduction in area due to epithelial contraction and migration. This treatment method benefited both ischemic and neuropathic lesions, regardless of their size, duration, anatomical location, type and progression of diabetes, and existing comorbidities. Table 2 summarizes the treatment method's effect. Regardless of the wounds' basic characteristics, all patients healed within a short period of approximately 60 days without adverse effects. Importantly, none of the wounds relapsed under this treatment method. None of the patients experienced a relapse by the six-month follow-up.None of these or other wounds that become chronic and are subject to primary or secondary refractory events would have healed without this treatment method in which effective co-administration with GHRP-6 and EGF infiltrations is made.

[0050] Table 2. Arrest of the healing process and impact of treatments. In the same clinical setting, another trial was conducted with 9 male patients for whom infiltrated EGF was contraindicated due to a history of prostate, skin, and lung tumors. Topical treatment with GHRP-6 at 400 pg / ml was applied only every other day after dressing changes. The lesions included lower leg ulcers and recurrent laryngeal artery disease (RLA) of both ischemic (3 patients) and neuropathic (6 patients) origin, all in the primary refractory phase. After 6 weeks of topical GHRP-6 administration in conjunction with standard treatment, no benefit was observed. This confirms that GHRP-6 alone does not effectively restart the arrested healing process.

[0051] Example 2. Demonstration of the “senotherapy” effect of the co-administration of EGF and GHRP-6.

[0052] To evaluate the anti-senescence effect of EGF and GHRP-6, both individually and in combination, biopsies were collected from the base and margins of complex wounds that had been present for more than 30 days and were refractory to various local and systemic medical interventions in five patients. These type II diabetic donors, with a disease duration of more than 15 years, were hospitalized and metabolically stable. The lesions were selected based on the criterion of being free of local infection. The collected biopsies were maintained in DMEM culture medium, enriched with 25% fetal bovine serum and a mixture of antibiotics and an antifungal agent, until processing in the laboratory. The tissue was meticulously fragmented and preserved in culture plates in DMEM+10% FBS at 37°C. After two weeks, primary fibroblast cultures were obtained from three patients.These cells were used to evaluate the presence or absence of molecular markers of senescence compared to human cutaneous fibroblasts from age-appropriate healthy donors undergoing cosmetic surgery. The parameters evaluated were (1) percent of p-Gal-positive cells, (2) percent of cells that incorporated and labeled with anti-bromodeoxyuridine (BrdU) antibody, (3) percent of cells with foci of senescence-associated heterochromatin (SAHF), (4) percent of cells with nuclear staining for p53 Ser-15, and (5) percent of cells positive for Cyclin D1 in the nucleus or cytoplasm. The presence or absence of each of these markers was evaluated according to a widely described and accepted methodology (de Zhuo X, Niu XH, Chen YC, Xin DQ, Guo YL, Mao ZB (2010) Vitamin D3 up-regulated protein 1 (VDUP1 ) is regulated by FOXO3A and miR-17-5p at the transcriptional and post-transcriptional levels, respectively, in senescent fibroblasts. J. Biol. Chem. 285, 31491-31501 ).Four experimental groups were organized, consisting of: (1) a control group of diabetic patient cells that did not receive treatment with the invention and were maintained in their culture medium; (2) a group of diabetic patient cells treated with 25 pg of EGF / ml of medium for 48 hours; (3) a group of diabetic patient cells treated with 25 pg of GHRP-6 / ml of medium for 48 hours; and (4) a group of diabetic patient cells treated with the combination of both agents at the aforementioned concentrations for 48 hours. Three replicates were used for each of the three patients (150,000 cells per patient). A reference control group consisting of cutaneous fibroblasts from healthy, non-diabetic donors was maintained without treatment and kept under the same culture conditions.

[0053] Table 3. Effect of EGF, GHRP-6 and their combination on markers of cellular senescence.

[0054] ** and *** p<0.001 one-way ANOVA

[0055] The results of these experiments demonstrate that GHRP-6 alone is capable of reversing phenotypic markers of cellular aging, such as the number of p-Gal-positive cells, the percentage of cells containing SAHF, and the percentage of cells with nuclear expression of p53-ser15. Notably, these cells show no response to the mitogenic stimulus of EGF, suggesting that the proliferative response to this growth factor occurs only after the senescent arrest scaffold has been reversed. This assertion is supported by the response of cells that were able to incorporate deoxyuridine (Brdll) and those that showed cyclin D1 expression. Although the effect of co-administration of both substances to diabetic fibroblasts does not correspond to the values ​​observed in healthy donor cells, it deviates significantly from the senile phenotype characteristic of basal conditions.

[0056] Example 3. Effect of topical treatment with GHRP-6 on lesions with arrest and primary refractoriness to infiltrative treatment with EGF.

[0057] Although infiltrated EGF has been a successful intervention for torpid, complex, and refractory wounds (Berlanga-Acosta J., et al. MEDICC Review, July 2020, Vol 22, No 3), some patients / wounds exhibit early refractoriness in responding to granulation tissue formation, which is the primary effect of the treatment. Infiltrated EGF does not always reverse the molecular factors of early chronicity and re-establish an uninterrupted healing trajectory. Consequently, we examined the effect of combined administration in a subgroup of seven hospitalized patients with atonic, non-granulated wounds who, despite standard medical interventions plus infiltrated EGF on alternate days, remained unresponsive from the outset, i.e., in primary refractoriness. Table 4 shows the characteristics of this study.

[0058] Table 4. Effect of co-administration on lesions with primary refractoriness to infiltrative treatment with EGF.

[0059] The results of this study demonstrate the ability of topically administered GHRP-6, in conjunction with local EGF infiltration, to reverse the primary refractory phase of complex lower limb lesions. This is a surprising finding with high clinical value for eliciting an early reparative response in the universe of peripheral soft tissue ulcers affecting humans.

[0060] Example 4. Topical application of GHRP-6 reverses re-chronification and promotes wound closure in patients treated with hyperbaric oxygen.

[0061] The study was conducted in a cohort of five diabetic patients hospitalized for high-grade lower extremity injuries. The wounds included residual bases of major and minor amputations (RAAs) of neuropathic and ischemic origin, classified as Wagner grades 3 and 4. Patients received a primary treatment regimen of hyperbaric oxygen therapy consisting of 100% oxygen at 2 atmospheres for 90 minutes every 24 hours for 21 days. All patients received standard care, including antiplatelet agents and advanced antibiotics, to control infection. Local wound care was performed, and dressings were applied daily before each immersion. Throughout the course of hyperbaric therapy, these injuries experienced disruption of the ongoing healing process, leading to arrest, chronicity, and consequently, secondary refractoriness.In this context, hyperbaric treatment was continued, and daily topical administration of GHRP-6 at 400 pg / ml in physiological saline solution was added. It was reproducibly demonstrated (Table 5) that the co-treatment regimen was again effective, triggering the resumption of the healing process until complete closure. It was shown that the topical application of GHRP-6 at 400 pg / ml can act synergistically with hyperbaric oxygenation, as observed for infiltrated EGF, in order to reverse a recurring chronic state.

[0062] Table 5. Effect of GHRP-6 treatment together with hyperbaric oxygenation on restarting the healing process.

[0063] Example 5. Topical application of GHRP-6 reverses re-chronification and promotes wound closure in patients treated with vacuum-assisted closure (VAC) machines.

[0064] A total of seven patients with lesions, including ulcers and residual amputation bases classified as Wagner grades 3 and 4 of neuropathic and ischemic origin, were successfully treated with different vacuum / negative pressure wound therapy regimens. Treatment began with a continuous-mode regimen applied for the first 48 hours, followed by intermittent pressure set at -125 mm Hg with pressure intervals of 5 minutes on and 2 minutes off. Most patients averaged more than 7 days of treatment with intermittent pressure changes every 3 to 5 days. Patients received standard care, including hypoglycemic agents, antiplatelet agents, and advanced antibiotics to control infection. Unexpectedly, some lesions began to halt the progressive healing process, resulting in a diagnosis of secondary refractory wound healing.The wounds were evaluated, and topical application of GHRP-6 at 400 pg / ml in physiological saline solution was added, maintaining the same intermittent regimen at -125 mm Hg with pressure intervals of 5 minutes on and 2 minutes off. With this treatment, the administration of GHRP-6 in conjunction with the negative pressure regimen achieved stable and progressive reversal of lesion healing, which progressed to complete epithelialization. It was demonstrated that the topical application of GHRP-6 at 400 pg / ml can act synergistically with vacuum therapy.

[0065] Table 6. Effect of co-application of negative pressure / vacuum therapy and GHRP-6 for reversal of refractoriness.

[0066] Example 6. Infiltrated EGF and topical application of GHRP-6 reverse chronicity or secondary refractoriness and promote closure of pressure lesions.

[0067] The study was conducted on a cohort of 10 patients with grade III and IV pressure injuries, starting from the initial stage of their primary injury. Patients were hospitalized and underwent postural repositioning. The EGF infiltration protocol was routinely applied unless otherwise indicated by specialists. These patients were included in the observational study because they experienced a sudden arrest of the healing process after their injuries had begun to progress satisfactorily and stably. All patients were receiving systemic antimicrobial therapy in addition to treatment for their underlying chronic conditions. Other palliative interventions were also implemented, including hydration management, analgesics, and postural repositioning. The wounds were debrided, cleaned with saline solution, and bandaged.Dressings and other local procedures were performed on alternate days. Once the wounds were clinically free of infection and the patients were stable, they were enrolled in the recombinant human EGF infiltration protocol, three times per week to accelerate healing. Accordingly, each lesion received 75 pg of EGF per treatment session (Fernandez-Montequin J. et al. Int Wound J. 2009 Dec;6(6):432-43. doi: 10.1111 / j.1742-481X.2009.00641.x). Three weeks after initiating EGF treatment, corresponding to 9 infiltration sessions, the lesions showed varying percentages of coverage with productive granulation tissue, encompassing approximately 30–50% of the damaged area. Treatments and wounds progressed satisfactorily until weeks 4–6, when some showed clinical signs of atonicity and wound edge arrest.Thus, granulation tissue growth, contraction, and epithelial migration were halted. These wounds were not infected, and analysis of the patients' medical records offered no evidence to elucidate the underlying cause of the halted healing. Table 7 reflects the main characteristics of the patients' pressure injuries, as well as the number of treatments with infiltrated EGF until the lesions reached a state of arrest. Lesions that showed primary refractoriness were examined and evaluated by specialists, defining the start of co-administration of the hexapeptide secretagogue GHRP-6 at a concentration of 400 pg / ml. The lesions were treated daily or every other day, always after undergoing local antisepsis followed by infiltration with epidermal growth factor.Again, co-administration of both agents was shown to reverse proliferative arrest in granulation tissue. As shown in Table 7, all lesions responded to co-administration of both agents, and the time considered as the restart of the healing process was between days 21 and 42.

[0068] Table 7. Characteristics of pressure injuries and EGF treatments up to the time of the scar arrest state.

[0069] (*) Represents the number of EGF infiltration sessions received up to the time of non-progression.

[0070] This intervention, performed for the first time in patients with pressure ulcers or bedsores who had suffered underlying head or spinal cord trauma, demonstrates the ability of co-administering a combination of epidermal growth factor infiltration and GHRP-6 to dismantle the molecular operators that lead to proliferative refractoriness to treatment with the former alone. As previously mentioned, although this GHRP-6 peptide does not show a proliferation-stimulating effect in cultured cells, it is capable of neutralizing the molecular operators that lead cells to proliferative arrest, as well as those that induce premature cellular senescence. This confirms that the pathophysiological basis of these refractory episodes lies in the interception of cellular arrest and senescence. Example 7.EGF infiltration and topical application of GHRP-6 reverse chronicity or secondary refractoriness and promote the closure of venous ulcers.

[0071] The study was conducted in a cohort of eight patients with full-thickness malleolar and supramalleolar venous ulcers. Patients were hospitalized. The EGF infiltration protocol was routinely applied unless otherwise indicated by specialists. These patients were included in the observational study because they experienced a sudden arrest of the healing process after their lesions had begun to evolve satisfactorily and stably. All patients were receiving systemic antimicrobial therapy in addition to treatment for their underlying chronic conditions. Other palliative interventions were also implemented, including hydration management, analgesics, offloading of the affected limb, and compression therapy with elastic bandages. The wounds were debrided, cleaned with saline solution, and bandaged.Dressings and other local procedures were performed every two days. Once the wounds were clinically free of infection and the patients were metabolically stable, they were enrolled in the infiltrated recombinant human EGF protocol, three times a week to accelerate closure. Accordingly, each lesion received 75 pg of EGF per treatment session (Fernandez-Montequin J. et al. Int Wound J. 2009 Dec;6(6):432-43. doi: 10.1111 / j.1742-481X.2009.00641.x).

[0072] Five weeks after initiating EGF treatment, corresponding to 15 infiltration sessions, the lesions showed varying percentages of coverage with productive granulation tissue, encompassing approximately 40% to 70% of the affected area. Treatments and wounds progressed satisfactorily until weeks 6 to 8, when some showed clinical signs of atonicity and wound arrest. This resulted in the cessation of granulation tissue growth, contraction, and epithelial migration. These wounds were not infected, and analysis of the patients' medical records provided no evidence to elucidate the underlying cause of the halted healing. Table 8 reflects the main characteristics of the patients' venous ulcers, as well as the number of EGF infiltration treatments until the lesions reached a state of arrest.

[0073] As with other wounds deemed refractory to treatment, the venous lesions were examined and evaluated by specialists, who determined the initiation of co-administration of the hexapeptide secretagogue GHRP-6 at a concentration of 400 pg / mL. Following the pre-established administration schedule, the lesions were treated daily or every other day, concurrently with infiltration of epidermal growth factor. Again, the co-administration of both agents was shown to reverse proliferative arrest in the granulation tissue. As shown in Table 8, all lesions responded to the co-administration of both agents, and the point at which the healing process restarted was between days 66 and 82.

[0074] Table 8. Characteristics of venous ulcers and EGF treatments up to the time of the scar arrest state.

[0075] (*) Represents the number of EGF infiltration sessions received up to the point of no progression. This intervention is also the first time it has been performed in patients with venous ulcers of different pathophysiological bases. In this type of chronic ulcer, the ability of co-administration between epidermal growth factor infiltration and GHRP-6 to reverse the episode of proliferative refractoriness triggered by treatment with the former is also demonstrated. Again, it is noteworthy that GHRP-6, at the dose and administration regimen used, appears to dismantle the mediators that lead to proliferative arrest or cellular senescence.

Claims

CLAIMS METHOD FOR THE TREATMENT OF SENESCENCE AND HEALING OF COMPLEX REFRACTORY CHRONIC WOUNDS 1. Method for preventing or reversing the phenomenon of premature senescence of skin cells and complex chronic wounds characterized by the application of an effective amount of the growth hormone secretagogue peptide GHRP-6.

2. Method according to claim 1 characterized in that the growth hormone secretagogue peptide GHRP-6 is applied in conjunction with a healing agent or therapy to eliminate primary or secondary refractoriness and restart the healing process of complex chronic wounds.

3. Method according to claim 2 characterized in that the secretagogue peptide is applied together with the same healing therapy at the time of the arrest of the healing process.

4. Method according to claim 2 characterized in that the healing agent or therapy consists of a growth factor, the application of hyperbaric oxygen or the application of a vacuum-assisted closure machine.

5. Method according to claim 4 characterized in that the growth factor is Epidermal Growth Factor (EGF).

6. Method according to claims 1 to 5 characterized in that the complex chronic wounds comprise diabetic foot ulcers, venous or varicose ulcers, pressure ulcers or radiation ulcers.

7. Method according to claims 1 to 5 characterized in that the growth hormone secretagogue peptide GHRP6 and the healing agent or therapy are administered sequentially or simultaneously by parenteral or topical route.

8. Use of the growth hormone secretagogue peptide GHRP-6 as an agent capable of preventing and reversing the phenomenon of premature senescence of skin cells and chronic wounds.

9. Use of the growth hormone secretagogue peptide GHRP-6 according to claim 8 in co-administration with a healing agent or therapy to eliminate primary or secondary refractoriness and restart the healing process of complex chronic wounds.

10. Use according to claim 9 wherein the secretagogue peptide is used in conjunction with the same healing therapy that was used when the arrest of the healing process occurred.

11. Use according to claim 9 wherein the healing agent consists of a growth factor, the application of hyperbaric oxygen, or the application of a vacuum-assisted closure machine.

12. Use according to claim 11 wherein the growth factor is Epidermal Growth Factor (EGF).

13. Use according to claims 8 to 12 wherein complex chronic wounds comprise diabetic foot ulcers, venous or varicose ulcers, pressure ulcers or radiation ulcers.

14. Use of the growth hormone secretagogue peptide GHRP-6 to eliminate primary or secondary refractoriness of complex chronic wounds.

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