Compositions and methods for promoting and / or maintaining vaginal, vulvar and perianal tissue vitality and tissue health - Patents.com

Abstract

The described invention provides a cosmetic composition formulated for topical application containing an aqueous gel component comprising hyaluronic acid, a plant raw material component comprising decarboxylated cannabidiol (CBD) representing about 20% of the isolate, and a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid, the composition being a slightly viscous, non-occlusive aqueous liquid, the finished product having a pH in the range of about 4.0 to about 5.0, and the composition being non-psychoactive. The described invention also provides a method for promoting and maintaining vaginal, vulvar, and perianal tissue vitality and tissue health in a subject in need thereof by topically administering the composition to the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to Provisional Application No. 63 / 239,755, filed September 1, 2021, the contents of which are incorporated by reference in their entirety.

[0002] The invention described relates to topical compositions, wound healing, tissue repair and rejuvenation. [Background technology]

[0003] Mechanisms of wound healing The term "wound healing" refers to the process by which the body repairs injury to any of its tissues, particularly injury caused by physical means and involving a break in continuity.

[0004] Wound healing response is often described as having three distinct phases: injury, inflammation, and repair.Generally speaking, the body responds to injury with an inflammatory response, which is essential to maintain the health and integrity of the organism.However, when this goes wrong, tissue destruction can occur.

[0005] Stage I: Disability Injuries caused by factors including, but not limited to, autoimmune or allergic reactions, environmental particulates, infections, or mechanical damage often result in the destruction of normal tissue architecture and initiate a healing response. Damaged epithelial and endothelial cells must be replaced to maintain barrier function and integrity, respectively, and to prevent blood loss. Acute injury to endothelial cells leads to the release of inflammatory mediators and the initiation of the antifibrinolytic coagulation cascade, temporarily plugging the damaged vessel with a platelet- and fibrin-rich clot.

[0006] Platelet recruitment, degranulation and clot formation rapidly progress to a stage of vasoconstriction accompanied by increased permeability, allowing extravasation (leukocytes move from capillaries into the surrounding tissue) and direct recruitment of leukocytes to the site of injury. The basement membrane forms an extracellular matrix underlying the epithelium and endothelium of parenchymal tissues, preventing direct access to the injured tissue. To disrupt this physical barrier, zinc-dependent endopeptidases, also called matrix metalloproteinases (MMPs), cleave one or more extracellular matrix components, allowing extravasation of cells to and from the site of injury. Specifically, MMP-2 (gelatinase A, type N collagenase) and MMP-9 (gelatinase B, type IV collagenase) cleave type N collagen and gelatin, two important components of the basement membrane. Recent studies have found that MMP-2 and MMP-9 are upregulated, highlighting that tissue destruction and regeneration processes are common in fibrotic conditions. The activity of MMPs is controlled by several mechanisms, including transcriptional regulation, proenzyme regulation, and specific tissue inhibitors of MMPs. The balance between MMPs and various inhibitory mechanisms can regulate inflammation and determine the net amount of collagen deposited during the healing response.

[0007] Phase II: Inflammation Once access to the site of tissue injury is achieved, chemokine gradients recruit inflammatory cells: neutrophils, eosinophils, lymphocytes, and macrophages are observed at the site of acute injury along with cellular debris and necrotic areas cleared by phagocytes.

[0008] Early recruitment of eosinophils, neutrophils, lymphocytes, and macrophages, which provide inflammatory cytokines and chemokines, may contribute to the accumulation of local TGF-β and IL-13. Following the initial injury and wave of inflammatory cells, late-stage recruitment of inflammatory cells may assist in phagocytosis, removal of cellular debris, and control of excessive cell proliferation, both of which may contribute to normal healing. Late-stage inflammation may play an antifibrotic role and may be required for successful resolution of the wound healing response. For example, a late-stage inflammatory profile enriched in phagocytic macrophages, which aid in fibroblast clearance, in addition to IL-10-secreting regulatory T cells, which suppress local chemokine production and TGF-β, may prevent excessive fibroblast activation.

[0009] The nature of the insult or causative agent often determines the nature of the inflammatory response that follows. For example, exogenous stimuli such as pathogen-associated molecular patterns (PAMPs) are recognized by pathogen recognition receptors such as toll-like receptors and NOD-like receptors (cytoplasmic proteins with various functions in regulating inflammatory and apoptotic responses) and influence the response of cells involved in the innate immune response to invasive pathogens. Endogenous danger signals can also influence local innate cells and orchestrate the inflammatory cascade.

[0010] The nature of the inflammatory response dramatically affects resident tissue cells and subsequent inflammatory cells. The inflammatory cells themselves also propagate further inflammation through the secretion of chemokines, cytokines, and growth factors. Many cytokines are involved throughout wound healing and the fibrotic response, and specific sets of genes are activated in various conditions. For example, chronic allergic airway disease in asthma is commonly associated with an elevated type 2 helper T cell (Th2)-associated cytokine profile, including but not limited to interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), and interleukin-9 (IL-9), whereas patients with chronic obstructive pulmonary disease and fibrotic lung diseases (such as idiopathic pulmonary fibrosis) more frequently exhibit a pro-inflammatory cytokine profile, including but not limited to interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and platelet-derived growth factor (PDGF). Each of these cytokines has been shown to exhibit significant pro-fibrotic activity, acting through the recruitment, activation, and proliferation of fibroblasts, macrophages, and myofibroblasts.

[0011] Phase III: Tissue repair and contraction The closure phase of wound healing consists of coordinated cellular reorganization, wound contraction, closure, and re-epithelialization induced by the formation of a scaffold rich in fibrin (a fibrous protein that polymerizes to form a "mesh" that forms a clot over the wound site). Most of the research elucidating the processes involved in this phase of wound repair has come from dermal wound studies and in vitro systems.

[0012] Collagen and smooth muscle actin (α-SMA) from myofibroblasts form a provisional extracellular matrix, and fibronectin from macrophages, platelets, and fibroblasts forms the fibrin scaffold. Collectively, these structures are commonly referred to as granulation tissue.

[0013] In addition to fibronectin, the provisional extracellular matrix consists of glycoproteins (such as PDGF), glycosaminoglycans (such as hyaluronic acid), proteoglycans, and elastin. Growth factors and TGF-β-activated fibroblasts migrate along the extracellular matrix network and repair the wound. Within skin wounds, TGF-β also induces a contractile response and regulates the orientation of collagen fibers. As mentioned above, differentiation of fibroblasts into myofibroblasts also generates stress fibers and neo-expression of α-SMA, both of which confer high contractile activity within the myofibroblasts. Attachment of myofibroblasts to the extracellular matrix at specialized sites called "fibronexus" or "super mature focal adhesions" pulls the wound together and reduces the size of the lesion during the contraction phase. The extent of extracellular matrix laid down and the amount of activated myofibroblasts determine the amount of collagen deposition. To this end, the balance of matrix metalloproteinases (MMPs) vs. tissue inhibitors of metalloproteinases (TIMPs) and collagen vs. collagenases changes throughout the response, shifting from pro-synthesis and increased collagen deposition to a regulated balance with no net increase in collagen. For successful wound healing, this balance often occurs as fibroblasts undergo apoptosis, inflammation begins to subside, and granulation tissue recedes, leaving behind a collagen-rich lesion.

[0014] Removal of inflammatory cells, especially α-SMA positive myofibroblasts, is essential to terminate collagen deposition. From skin studies, re-epithelialization of wound sites re-establishes barrier function and allows encapsulated cell reorganization. Several in vitro and in vivo models have been used to identify key stages of cell migration, proliferation, and cell spreading using human or rat epithelial cells grown on collagen matrices, or tracheal wounds in vivo. Rapid and dynamic motility and proliferation that accompanies epithelial repair from the edge of the denuded area occurs within hours of the initial wound. Furthermore, sliding sheets of epithelial cells can migrate over the wounded area and assist in wound coverage. Several factors have been shown to regulate re-epithelialization, including serum-derived transforming growth factor alpha (TGF-α) and matrix met alloproteinase-7 (MMP-7), itself regulated by TIMP-1.

[0015] Overall, the degree of inflammation, angiogenesis, and amount of extracellular matrix deposition all contribute to the ultimate development of a fibrotic lesion or scar. Thus, therapeutic interventions that interfere with fibroblast activation, proliferation, or apoptosis require understanding and evaluation of all stages of wound repair. Although these three phases are often presented sequentially, during chronic or repetitive injury, these processes function in parallel, placing great demands on regulatory mechanisms. (Wilson and Wynn, Mucosal Immunol., 2009, 3(2):103-121).

[0016] Skin symptoms of hemorrhoids One in three Americans has hemorrhoids at screening colonoscopy [Sandler, RS and Peery, AF, Clin. Gastroenterol. Hepatol. (2019) 17(1):8-15, citing Everhart, JE and Ruhl, CE. Gastroenterology (2009) 136:741-54]. Despite their prevalence and increasing treatment, symptomatic hemorrhoids remain poorly understood with little evidence to guide treatment.

[0017] Hemorrhoids are clusters of vascular tissue, smooth muscle, and connective tissue arranged in three rows along the anal canal. [Ibid., citing Sun, Z. and Migaly, J. Clin. Colon. Rectal Surg. (2016) 29:22-9] They exist in healthy individuals as cushions to help maintain drainage. [Ibid., citing Ganz, RA. Clin. Gastroenterol. Hepatol. (2013) 11:593-603] Although hemorrhoids are normal structures. [Ibid., citing Haas, PA, et al. Dis. Colon Rectum (1984) 27:442-50] The term hemorrhoid has come to refer to a pathological or symptomatic process. [Ibid., citing Sun, Z & Migaly, J. Clin. Colon Rectal Surg. (2016) 29:22-9]

[0018] Internal hemorrhoids, located above the dentate line, are covered by columnar epithelium innervated by visceral nerve fibers not associated with pain. Internal hemorrhoids are graded based on the degree of prolapse.

[0019] External hemorrhoids lie below the dentate line. They are covered by squamous epithelium and innervated by somatic nerves that can cause pain. External hemorrhoids are generally asymptomatic unless they are thrombotic. Thrombosed hemorrhoids are extremely painful. [Ibid., citing Madoff, RD and Fleshman, JW. Gastroenterology (2004) 126:1463-73]. Once external hemorrhoids resolve, the skin tag may persist and become irritating or cause hygiene problems.

[0020] According to Goligher's classification, the severity of hemorrhoids is classified into four grades. Hemorrhoids are classified based on the degree of prolapse. Grade I, do not prolapse below the dentate line and can be seen on anoscopy or colonoscopy. Grade II, prolapse below the dentate line but shrink spontaneously. Grade III, prolapse, requires manual reduction. Grade IV, prolapse and remain below the dentate line. They are not reducible.

[0021] The etiology of hemorrhoids is unclear. Hemorrhoids are commonly reported in women, primarily during pregnancy and after vaginal delivery. It is generally believed that pregnancy and vaginal delivery predispose women to developing symptomatic hemorrhoids for several reasons, including hormonal changes, increased intra-abdominal pressure, straining during bowel movements due to constipation, prolonged straining for more than 20 minutes during the second stage of labor, and delivery of a baby weighing more than 3800 g [Kestranek, J. Drugs in Context (2019) 8: 212602]. However, one report found that overweight and pregnancy were not associated with current hemorrhoids [ibid. citing Peery, AF et al. PLoS One (2015) 10: e0139100]. Others found an association with BMI but no association with age or pregnancy [Ibid. citing Riss, S. et al. Intl J. Colorectal Disease (2011) 27:215-220].

[0022] Instructions for patients published in JAMA read: "Anything that puts pressure on the veins in the lower body can lead to hemorrhoids, including straining during bowel movements, sitting on the toilet for long periods of time, constipation or diarrhea, being overweight, pregnancy, and aging, which weakens the tissue." [Sandler, RS and Peery, AF, Clin. Gastroenterol. Hepatol. (2019) 17(1):8-15, citing Sugarman, DT. JAMA (2014) 312:2698].

[0023] Symptoms due to hemorrhoids include bleeding, pain, itching, fecal leakage, prolapse, and mucus discharge. [Ibid. citing Ganz, RA. Clin. Gastroentrol. Hepatol. (2013) 11:593-603] However, a 2001 study from Germany looking at 458 patients with abdominal and / or anal symptoms supports the idea that symptoms associated with hemorrhoids may have other causes.

[0024] Treatments for hemorrhoids include medical therapy, non-surgical office-based treatments, and surgery. [Id., citing Madoff, RD and Fleshman, JW. Gastroenterology (2004) 126:1463-73].

[0025] Dietary and lifestyle changes, which require high patient compliance, are usually considered the first step of a conservative strategy. However, the evidence supporting these interventions is anecdotal [Kestranek, J. Drugs in Context (2019) 8: 212602]. Since inflammation plays an important role, especially in the cutaneous manifestations of hemorrhoidal disease [ibid. citing Abramowitz, L. et al. Aliment. Pharmacol. Ther. (2010); 31 (Suppl. 1) 1-58], treatment is mainly based on the use of topical preparations containing anti-inflammatory drugs, including steroids, anesthetics, astringents and / or antiseptics [ibid. citing Davis, BR et al. Dis. Colon Rectum. (2018) 61: 284-92]. However, in some cases, e.g. steroids, are associated with the potential development of adverse events, and in most cases no randomized trials have been conducted to evaluate the efficacy and safety of different interventions. [Id., citing Brown, SR. Ther. Adv. Chronic Dis. (2017) 8(10):141-47, Davis, BR et al. Dis. Colon Rectum (2018) 61:284-92].

[0026] Some commonly used combinations include ketocaine / fluocinolone and hydrocortisone / benzocaine. Corticosteroids may be effective in this scenario, but these molecules, often available as prodrugs, may be associated with the risk of systemic absorption, distribution, metabolism, and excretion. Thus, their higher lipophilicity may limit their application over intermediate periods or in elderly, lactating, or pregnant women.

[0027] The topical combination of tribenoside and lidocaine (sold under the brand Procto-Glyvenol®, Recordati SpA, Italy) is a pharmaceutical formulation for the local treatment of hemorrhoids that has been used for decades to treat hemorrhoids in patients of either gender [Lorenc, Z. Gokce, O. Eur. Review Medical & Pharmacological Sciences (2016) 20:2742-51]. It is delivered as a suppository or a rectal cream. Its efficacy and safety are supported by several well-conducted studies and extensive clinical experience. This product combines the rapid local anesthetic action of lidocaine (to provide rapid relief of pain and itch) with the efficacy of tribenoside (a sugar derivative that is reported to interact with epidermal cells and modulate the expression and localization of laminin, thus aiding in the remodeling of the basement membrane in hemorrhoidal wound healing) to reduce inflammation, promote local healing and facilitate the restoration of local vascularity to its normal state. This dual mechanism of action purportedly allows for control of both the subjective (eg pain and discomfort) and objective (eg prolapse and bleeding) symptoms of hemorrhoids.

[0028] Amazon.com lists over 100 hemorrhoid treatments, many of which claim benefits or have questionable ingredients. [Sandler, RS and Peery, AF, Clin. Gastroenterol. Hepatol. (2019) 17(1):8-15]:

[0029] Therefore, a topical formulation that provides an alternative to corticosteroids but still has anti-inflammatory and wound healing effects would be highly desirable.

[0030] Sexual Side Effects of Cancer Treatment Radiation to the pelvic area can affect a woman's sexual health during and after treatment because the radiation beams damage the delicate tissues in and around the female genitals. Certain types of cancer are treated with radiation implants, which are radiation sources that are placed in the bladder, uterus, or vagina for a set number of days. During treatment, the tissues in the treatment area may become irritated and may become pink, swollen, and appear sunburned. A woman's vagina may feel tender during radiation treatment and for several weeks after. Radiation to the vagina can also damage the lining, making it thin and fragile. In rare cases, vaginal ulcers or open sores may develop and may take several months to heal after radiation therapy has ended. Once treatment has ended and the irritation has healed, scarring may occur and the walls of the vagina may become leathery and tough. Radiation treatment can also shorten or narrow the vagina, meaning the walls may not stretch as much during sex, which can cause pain. In some cases, the bladder and bowels are damaged, which can also affect sexual health.

[0031] Many of these side effects become chronic conditions that tend to worsen over the years, therefore prompt and long-term treatment is necessary to achieve good results and avoid recurrence of symptoms when treatment is stopped.

[0032] Women at highest risk of sexual side effects of cancer treatment include those being treated for bladder, breast, gynecological cancers (e.g., cervical, endometrial, ovarian, vaginal, vulvar), colon, rectal, and uterine cancer.

[0033] breast cancer As mortality rates decline, patients who survive breast cancer require ongoing management of the sequelae from the disease or its treatment. Breast cancer survivors may have a variety of symptoms associated with low estrogen levels, as a result of chemotherapy-induced ovarian failure or anti-estrogen hormone therapy, including, but not limited to, hot flashes, dyspareunia, vaginal dryness, and urogenital atrophy.

[0034] Genitourinary atrophy is a sign of estrogen deficiency in breast cancer patients undergoing chemotherapy or endocrine therapy and can induce various symptoms in the vulvovaginal area, including vaginal dryness, burning, itching, dyspareunia, and abnormal secretions. Lee, YK et al. Obstet. Gynecol. (2011) 117: 922-7]. Symptoms of urogenital atrophy are common in breast cancer survivors, but their optimal management remains unknown. Estrogen replacement may resolve urogenital symptoms, but is contraindicated in breast cancer patients, and even topical administration of estradiol tablets may affect serum hormone levels. [Ibid., V. Lancet (2003) 362: 419-27, citing Rosenberg, LU et al. Breast Cancer Res. (2006) 8: R11].

[0035] Chemotherapy drugs generally irritate all mucous membranes in the body, including the vulva and vaginal tissues. Inflammation can cause burning and irritation. Drugs that affect hormone production and absorption, such as tamoxifen and various aromatase inhibitors, also alter the body's use of estrogen, causing vaginal health problems.

[0036] In case of vaginal dryness and dyspareunia, guidelines recommend the use of vaginal lubricants for intercourse and vaginal moisturizers for general comfort. [Zoberi, K. & Tucker J. Am. Fam. Physician (2019) 99 (6): 370-75]. A systematic review of randomized controlled trials (RCTs) evaluating treatment options for urogenital atrophy in breast cancer patients is reported in Mazzarelio, S. et al. Breast Cancer Res. Treat. (2015) 152 (1): 1-8. An electronic literature search was performed for relevant citations from EMBASE, Ovid Medline and the Cochrane Library from 1946 to November 2014. No restrictions were placed on disease status (early vs. metastatic), patient age, and previous anticancer treatment. Interventions included pH-balancing gel, Replens® (vaginal moisturizer containing polycarbophil, mineral oil, hydrogenated palm oil glycerides, glycerin, carbomer homopolymer type B, sodium hydroxide, and sorbic acid), lidocaine, Estring® (estradiol vaginal ring containing silicone polymer and barium sulfate), and Vagifem® (film-coated tablet containing 10 μg estradiol as estradiol hemihydrate, hypromellose, lactose monohydrate, corn starch, and magnesium stearate). All doses, preparations, and dosing frequencies were considered. Outcomes included improvements in both vaginal symptoms (e.g., dryness, pain, dyspareunia, and itching) and vaginal hormonal responses measured by validated scales [e.g., Vaginal Health Index (VHI) and Vaginal Maturity Index (VMI)]. Of 430 unique citations identified, 4 studies (n=196) met the inclusion criteria. Sample sizes ranged from 7 to 98 patients. Given the heterogeneity of the studies, a narrative synthesis of the results was performed using the VHI score.One study of 98 patients suggested that vaginal pH balance gel (mean VHI 5.00 ± 0.816, mean VMI 51.18 ± 3.753) was more effective at relieving vaginal symptoms than placebo (VHI 16.98 ± 3.875, p < 0.001, VMI 47.87 ± 2.728, p < 0.001) at 12 weeks. In patients who used lidocaine, 90% had reduced dyspareunia compared to saline in a study of 46 patients. Although increases in serum estradiol occurred, a study of 7 patients showed that both Estring® and Vagifem® improved quality of life and VMI.

[0037] Gynecologic cancer Treatment of gynecologic malignancies is associated with many causes of genitopelvic pain and dyspareunia. Many of these symptoms result from changes in vaginal health due to surgery, radiation, and chemotherapy, which can cause vaginal shortening, narrowing, atrophy, and dryness.

[0038] endometrial cancer Endometrial cancer is the most common gynecologic malignancy in Western countries. The majority occurs in postmenopausal women, and surgery is the primary treatment for most patients. Even in the absence of adjuvant therapy in the form of radiation therapy and / or chemotherapy, patients are at risk of experiencing sexual dysfunction. [Huffman,LB e al.Gynecol.Oncol.(2016)140(2):359-68] A prospective evaluation (N=72) of the prevalence of sexual dysfunction in patients with early stage (I-IIIa) endometrial cancer 1-5 years after initial surgical treatment showed that 89% of participants had some form of sexual dysfunction as determined by a Female Sexual Function Scale (FSFI) score <26, with pain being the most commonly affected area. Only 18% of participants received adjuvant radiation therapy. Adjuvant therapy in the form of radiation therapy and / or chemotherapy is typically recommended for patients with a high risk of recurrence and high stage of disease. The Postoperative Radiotherapy in Endometrial Cancer (PORTEC-2) study investigated the outcomes and adverse effects of vaginal brachytherapy (VBT) compared with external beam radiotherapy (EBRT) for the treatment of high-intermediate risk endometrial cancer. [Ibid., citing Nout, RA et al. The Lancet (2010) 375:816-23]. There was no difference in sexual function between VBT and EBRT patients, but participants in the study reported significantly more vaginal dryness and less sexual interest, activity, and enjoyment when compared with an age-matched control population. [Ibid., citing Nout, RA et al. Eur. J. Cancer (2012) 48(11):1638-48]. In another study, vaginal changes after radiation included vaginal stenosis (meaning narrowing), vaginal scarring, mucosal telangiectasia (meaning dilation of previously existing small blood vessels or terminal blood vessels located near the surface of the mucosa, commonly known as spider veins), and mucosal atrophy (meaning thinning, dryness, and inflammation of the vaginal wall that can occur when there is little estrogen in the body) [Ibid. citing Nunns, DJ Gynecological Cancer (2000) 10(3):233-38].

[0039] cervical cancer Surgical treatment of early stage cervical cancer may include cervical conization, simple hysterectomy, or radical hysterectomy with pelvic lymphadenectomy. Radical hysterectomy is associated with adverse effects on sexual health and quality of life [Ibid., citing Greimel ER, et al. Psychoncology. (2009) 18(5):476-82]. Persistent sexual health concerns include lack of sexual interest (25%), lymphedema (19%), genital numbness (71%), and lack of lubrication (24%). [Ibid., Jensen PT, et al. Cancer. (2004) 100(1):97-10625-27, Pieterse, QD et al. Int J Gynecol Cancer. (2013) 23(9): 1717-25, Pieterse, QD et al. Int J Gynecol Cancer.(2006)16:1119-1129].

[0040] Radiation therapy in the form of EBRT and VBT, with or without concurrent chemotherapy (chemoradiotherapy), plays a major role in the treatment of cervical cancer in both primary and adjuvant settings. It is associated with major vaginal toxicities, including stenosis, shortening, atrophy, fibrosis, and dyspareunia (ibid. Katz, A. et al. Int J Gynecol Cancer. (2001) 11:234-35; Schover, LR, et al., Cancer. (1989) 63:204-212; Bergmark, K. et al. N Engl J Med. (1999) 340(18):1383-89; Brand AH, et al. Int J Gynecol Cancer. (2006) 16(1):288-93; Bruner DW, et al. Int J Radiat Oncol Biol Phys. (1993) 27:825-830). Primary or adjuvant radiation therapy is associated with greater sexual dysfunction and vaginal toxicity compared with surgery alone. [Ibid., Greimel ER, et al. Psychoncology. (2009) 18(5):476-82, citing Frumovitz M, et al. J Clin Oncol. (2005) 23(30):7428-36]. Compared with age-matched controls, cervical cancer patients treated with radiation had significantly more sexual dysfunction and vaginal morbidity, including decreased libido (85%), dissatisfaction with sex life (30%), decreased vaginal size (50%), dyspareunia (55%), and lack of lubrication (35%). [Ibid., citing Jensen PT, et al. Intl J. Radiation Oncology*Biology*Physics. (2003) 56(4):937-949]. The majority of patients with dyspareunia and lack of lubrication were distressed by their symptoms. [Ibid., citing Jensen PT, et al. Intl J. Radiation Oncology*Biology*Physics. (2003) 56(4):937-949].

[0041] Ovarian cancer Primary treatment for ovarian cancer typically consists of a series of surgeries and chemotherapy. Surgery includes hysterectomy, lymphadenectomy, and cytoreduction, with the goal of optimal cytoreduction before and after chemotherapy. Removal of the ovaries results in hormonal changes that may cause adverse changes in sexual health. [Ibid., Hughes C,et al.,Gynecologic Oncology.(1991)40:42-45]. Menopausal symptoms caused by cancer treatment may be more abrupt, prolonged, and intense [Ibid., Schover LR.J Clin Oncol.(2008)26(5):753-8], and if unmanaged, may lead to decreased quality of life, function, and sexual desire. [Ibid., Krychman ML,et al.,Oncology(2006)71(1-2):18-25]. Compared to healthy women, ovarian cancer survivors report increased vaginal dryness, more dyspareunia, less sexual activity, and lower libido. [Ibid., in Liavaag AH,et al.Gynecol Oncol.(2008)108(2):348-54,54]. In one study, sexual function in ovarian cancer patients was investigated based on treatment modality, comparing surgery alone in patients with early stage ovarian cancer (group 1), surgery combined with chemotherapy (group 2), and advanced inoperable or metastatic ovarian cancer patients receiving chemotherapy alone (group 3). Sexual satisfaction decreased in all patients after treatment, but was more pronounced in groups 2 and 3 [Bukovic D,et al.(2008)26(2):63-73].

[0042] vulvar cancer Treatment of vulvar cancer consists of primary surgery with or without adjuvant or primary radiation therapy based on size, location, and suspicion of lymph node involvement [Ibid., citing Stehman FB & Look KY. Obstet Gynecol. (2006) 107(3):719-33]. Surgical treatment has evolved from a radical "en bloc" resection of the vulva with bilateral inguinal and pelvic lymphadenectomy to a triple incision technique without pelvic lymphadenectomy [Ibid., citing Hacker, NF et al. Obstet Gynecol. (1981) 58:574-79,61]. Despite changes in surgical approaches, sexual morbidity remains prevalent. Physical changes after surgery may include vaginal narrowing, numbness along the scar, removal of the clitoris, and changes in tissue quality [ibid., citing Barlow EL, et al. J Adv Nurs. (2014) 70(8):1856-66, Janda M, et al. Int J Gynecol Cancer. (2004) 14:875-88166, Weijmar Schulz W, et al. Cancer (1990) 66:402-407].

[0043] Radiation therapy has a variety of roles in the treatment of vulvar cancer. In the adjuvant setting, radiation therapy can be administered to the vulva to treat positive or closed surgical margins, and to the groin and pelvis in the setting of positive lymph nodes to prevent recurrence and improve survival. Definitive chemoradiation therapy is recommended in advanced vulvar cancer that is not amenable to surgical resection.

[0044] There is a paucity of studies evaluating sexual health after radiation in patients with vulvar cancer. A longitudinal study observed a significant decrease in arousal and ability to induce sexual activity, as well as a decrease in perception of positive genital sensations, with or without adjuvant radiation 6 months after surgery, which did not improve during a 2-year follow-up [Ibid., citing Weijmar Schulz W, et al. Cancer (1990) 66:402-407].

[0045] Genitourinary syndrome of menopause (GSM) Genitourinary syndrome of menopause (GSM) is an accepted term to describe urogenital symptoms and signs associated with menopause, such as dryness, burning, irritation, and discomfort or pain, as well as sexual symptoms such as sexual dysfunction [Alvisi, S. et al. Medicina (2019) 55(10):615]. This condition, which includes vulvovaginal atrophy (VVA), can be accompanied by urinary signs and symptoms, including urinary incontinence, painful urination (dysuria), slow and painful urination (stranglerism), and frequent urinary tract infections [ibid. citing Gandhi, J. et al. Am. J. Obstet. Gynecol. (2016) 215:704-11]. Recent studies suggest that GSM affects most menopausal and postmenopausal women, with prevalence ranging from 36% to nearly 90%. The condition is already present in the premenopausal years, with a reported prevalence of 19% in women aged 40-45 years. [Ibid.] Despite its high prevalence, it remains underdiagnosed and undertreated.

[0046] The decline in circulating hormone levels, especially estrogen, represents the main trigger that determines vulvovaginal atrophy. The vaginal epithelium of postmenopausal women shows a flat epithelial surface characterized by keratinization and the absence of papillae. There are multiple layers of parabasal cells with a high nucleoplasmic to cytoplasmic ratio and almost no intermediate and superficial cells with reduced glycogen stores. This leads to a decrease in the number of Lactobacilli, resulting in an increase in vaginal pH [Miller, EA et al. Front. Microbiol. (2016) 7: 1936]. The low proportion of Lactobacilli and the increased relative proportion of anaerobic bacteria found in postmenopausal women may predispose to symptomatic VVA, although not all studies have consistently reported this association [ibid., citing Hummelen, R. et al., PLoS ONE (2011) 6: e26602, Brotman, RM et al. Menopause (2014) 21: 450, Shen, J. et al. Sci. Rep. (2016) 6: 24380].

[0047] A hypoestrogenous vaginal state also typically involves changes in connective tissue composition with a decrease in the type I / III collagen ratio, which results in decreased tissue strength [Ibid., citing Hulmes, DJ S. J. Struct. Biol. (2002) 137:2-10]. Thinning of the vaginal epithelium increases susceptibility to trauma, resulting in bleeding, spotting, and ulceration with any type of pressure, including sexual activity or simple gynecological manipulation. Thinning also exposes the underlying connective tissue, which is more vulnerable to inflammation or infection.

[0048] Due to these histological changes, clinical signs at the vaginal level include dryness and inadequate hydration, redness, loss of elasticity, petechiae, ulceration, inflammation, atypical discharge, fibrosis and vaginal obstruction. [Ibid.] At the vulva level, the most frequent signs include reduced tissue thickness, cohesion of the labia, loss of pubic hair and scratching due to itching. The resulting symptoms include vaginal dryness and superficial dyspareunia, with a prevalence of 78% and 76%, respectively [Ibid., citing Nappi, RE et al. Climacteric. (2016) 19:188-97], which may be associated with itching, burning and sensitivity to mechanical injury, leucorrhea or atypical discharge. At the vulvar level, the most frequent symptoms are burning, pain, increased sensitivity to physical and chemical irritants, and mechanical damage [Ibid. citing Murina, F. et al. Gynecol. Endocrinol. (2018) 34:631-35].

[0049] Women's sexuality and relationships are greatly affected by these changes [Ibid., citing Leiblum, S. et al. JAMA (1983) 249:2195-98]. The REVIVE study suggested that VVA symptoms significantly impacted patients' ability to achieve pleasurable intimate relationships (74%) and spontaneity (70%). 75% of sexually active postmenopausal women with VVA reported significantly decreased libido as a direct result of symptoms associated with the condition [Ibid., citing Nappi, RE et al. Climacteric. (2016) 19:188-97]. A 2014 study showed that most women were concerned that vaginal discomfort could have a long-term impact on their relationships [Ibid., citing Simons, JA et al. Menopause (2014) 21:137-42].

[0050] diabetes Chronic hyperglycemia associated with diabetes can lead to end-organ dysfunction and failure that may involve the retina, kidney, nerves, heart, and blood vessels. [Tsalamandris, S. et al. Eur. Cardiol. (2019) 14(1):50-59, citing Inzucchi, SE. N. Eng. J. Med. (2013) 368:193]

[0051] Diabetes is an important cause of sexual dysfunction in both men and women. In men, diabetic autonomic neuropathy, often reinforced by antihypertensive drugs and vascular disorders, is the main cause of organic impotence [Enzlin, P. et al. Diabetic Medicine (1998) 15: 809-15], and in diabetic women, problems affecting the vaginal canal are very often ignored. In fact, the literature on female sexual dysfunction and diabetes is limited, perhaps because of the tendency to ignore the sexual impact of women's physical illnesses or because of the difficulties posed by the complexity of the female sexual response [Enzlin, P. et al. Diabetic Medicine (1998) 15: 809-15, Gupta, L. et al. Touch Medical Media (2018)].

[0052] Sexual dysfunction in women is associated with both type 1 and type 2 diabetes [Enzlin, P. et al. Diabetes Care (2009) 32 (5): 780-5], and the risk of sexual dysfunction is more than 2.47 times higher in diabetic women [Barnard, KD et al. Practical Diabetes (2019) 36.5]. For example, women with diabetes are at high risk of recurrent vaginitis caused by Candida spp., which leads to inflammation resulting in dyspareunia and cystitis. These problems are often associated with vaginal dryness, redness, burning and itching during intercourse [Carati, D. et al. Clin. Exp. Obstet. Gynecol. Doi: 10.12891 / ceog3078.2016]. In diabetic women, Candida albicans binds more readily to epithelial cells, and hyperglycemia may affect humoral responses, leading to decreased neutrophils, chemotaxis, and phagocytosis. (Id. Segal, E. et al. J. Med. Vet. Mycol. (1984) 22:191, citing Bagdade, JD et al., Diabetes (1974) 23:9.) Vaginal dryness is twice as likely in diabetic women compared to nondiabetic women. (Enzlin, P. et al., Diabetes Care (2002) 25(4):672-77, citing Enzlin, P. et al. Diabet. Med. (1998) 15:809-15.)

[0053] The emerging role of inflammation in the pathophysiology of both type 1 and type 2 diabetes (T1D and T1D) and associated metabolic disorders has led to increased interest in targeting inflammation to improve disease prevention and control [Tsalamandris, S. et al. Eur. Cardiol. (2019) 14(1):50-59].

[0054] Treatment options Treatment options for women with sexual side effects conditions range from moisturizers to medications to energy-based devices. Despite the many types of treatments available, women are not satisfied with the options for a variety of reasons, and alternative therapies using moisturizers or lubricants do not provide relief for their symptoms.

[0055] A variety of non-hormonal non-prescription treatments for vaginal atrophy (VA) exist, including but not limited to increased sexual activity, smoking cessation, pelvic floor physical therapy (PT), and lubricants or moisturizers [Alvisi, S. et al. Medicina (2019) 55(10):615], citing Leiblum, S. et al. JAMA (1983) 249:2195-98]. Many women use over-the-counter (OTC) products such as vaginal lubricants and moisturizers. International guidelines consider these to be the first line of treatment in the treatment of VVA, with no significant contraindications and side effects [ibid., citing Stuenkel, CA et al. Clin. Endocrinol. Metab. (2015) 100:3975-4011]. These can be used alone or in combination with hormonal therapy if necessary. This treatment option is also recommended for women who cannot tolerate the use of vaginal estrogen preparations. The osmolality, pH, and composition of these products, either lubricants or moisturizers, should be similar to vaginal secretions [Ibid., citing Edwards, D., Panay, N. Climacteric. (2016) 19:151-61].

[0056] The main difference between vaginal lubricants and moisturizers is the timing of application. Vaginal lubricants are particularly suitable for women whose main concern is vaginal dryness during intercourse. Lubricants provide short-term relief of dryness and reduce difficult or painful intercourse (dyspareunia). They can be water-based, which are water-soluble and tend to dry out, oil-based, which are more durable but have a less lubricating effect, or silicone-based. Some lubricants contain glycerin, propylene glycol, sweeteners, and parabens, which can affect the pH and osmolality of water-based products [ibid., citing Edwards, D., Panay, N. Climacteric. (2016) 19:151-61].

[0057] Vaginal moisturizers are insoluble hydrophilic cross-linked polymers with characteristic bioadhesive properties that allow them to attach to the epithelium of the vaginal wall by retaining water. They can also contain large amounts of excipients that affect the pH and osmolality of the formulation. They can be used more regularly, not just in conjunction with sexual activity, and have a long-term effect of improving moisture of the vaginal mucosa and lowering the pH. The frequency of use is directly proportional to the severity of VVA [ibid., citing Edwards, D., Panay, N. Climacteric. (2016) 19: 151-61]. The acute application is a topical application in the evening before bedtime, which can be applied for 7 to 10 consecutive days to allow it to act overnight, followed by topical applications twice a week to maintain the beneficial effects. The most commonly used moisturizers are based on hyaluronic acid (HA), a glycosaminoglycan produced by fibroblasts, the main component of the extracellular matrix. A possible mechanism of action of hyaluronic acid is cell migration, as it has a very high capacity to bind water, which may promote cell migration [Salwowska, NMK J. Cosmet. Dermatol. (2016) 15: 520-26]. In the case of tissue damage, it has been suggested that HA may stimulate fibroblast migration and proliferation, and thus collagen fiber deposition, in addition to stimulating neovascularization and re-epithelialization. When used regularly, daily or every 2-3 days, HA-based products improve symptoms of vaginal dryness, and the effects have been compared to those of topical estrogen therapy [ibid., citing Mitchell, CM et al. Menopause (2019) 26: 816-22]. Several adverse effects have been reported with the use of HA [ibid., citing Chung, KL et al. Aesthet. Surg. J. (2019) doi:10.1093 / asj / sjz222]; most occur after injection. These include local reactions, i.e., bruising, erythema, swelling, and, rarely, more severe events such as tissue necrosis, infection, or pulmonary complications. To our knowledge, no serious adverse effects have been reported with the use of HA-based vaginal moisturizers. [ibid.]

[0058] Other possible components of vaginal moisturizers are ozonides, intermediate products of ozone, which act as biological reservoirs that maintain the therapeutic power of the molecule. Upon contact with living tissue, ozonides are rapidly activated and stimulate local microcirculation to induce neovascularization, promote tissue repair, and inhibit pro-inflammatory prostaglandins [Ibid., citing DiMauro, R. et al. Int. J. Mol. Sci. (2019) 20:634].

[0059] Oral vitamin D and vaginal vitamin E have been proposed for the treatment of VVA, but efficacy data are limited and sometimes inconsistent. Vitamin D stimulates proliferation of the vaginal epithelium by activating the vitamin D receptor (VDR). Vaginal vitamin E is involved in the metabolism of all cells and prevents tissue damage caused by oxidants. This promotes blood circulation and, as a result, increases the metabolism of the vaginal connective tissue and increases moisture and flexibility of the vaginal wall [ibid., Yildirim, B. et al. Maturitas (2004) 49:334, Pitsouni, E. et al. Eur. J. Obstet. Gynecol. Reprod. Bio. (2018) 229:45-56, citing Costantino, D. & Guaraldi, C. Eur. Rev. Med. Pharmacol. Sci. (2008) 12:411].

[0060] Oral and intravaginal probiotics to improve the vaginal microbiota may be beneficial in treating VVA symptoms, but placebo-controlled trials demonstrating their efficacy are lacking [ibid. citing Muhleisen, AL & Herbst-Kralovetz, MM. Maturitas (2016) 91:42-50].

[0061] Oral phytoestrogens are not effective [Ibid. citing Grant, MD et al. AHRQ Comparative Effectiveness Reviews. Agency for Healthcare Research and Quality (US); Rockville, MD, USA: 2015. Menopausal Symptoms: Comparative. Effectiveness of Treatments.] Preliminary studies suggest that topical phytoestrogens may have beneficial effects on VVA, improving genital symptoms, maturation index, vaginal pH, morphology, and expression of estrogen receptors in the vaginal epithelium [Ibid. citing Kagan, R. et al. Menopause (2010) 17:281-89].

[0062] Hormone therapy for menopause (HTM), meaning estrogen-progestin, estrogen-bazedoxifene, tibolone, or exclusively estrogen-related, in women who have had a hysterectomy, has beneficial effects on many symptoms associated with menopause, including VVA. According to international guidelines, it is not recommended for women suffering from vaginal and vulvar symptoms only, but evidence shows that when used for the primary indication, HTM can restore physiological vaginal pH, maturation index, and thickness of the vaginal epithelium, its vascularization, and lubrication [ibid., citing The NAMS 2017 Hormone Therapy Position Statement Advisory Panel The 2017 hormone therapy position statement of the North American Menopause Society.Menopause.2017;24:728-753.doi:10.1097 / GME.0000000000000921].

[0063] International guidelines recommend topical hormone therapy as a second line treatment if vaginal lubricants and moisturizers are ineffective [Ibid., citing The NAMS 2017 Hormone Therapy Position Statement Advisory Panel The 2017 hormone therapy position statement of the North American Menopause Society.Menopause.2017;24:728-753.doi:10.1097 / GME.0000000000000921]. Available options include estradiol, estriol, conjugated estrogens or promestriene gels, creams, ovules, tablets, or rings. These are specifically indicated for the treatment of VVA, including dyspareunia. All estrogen-based vaginal products are more effective than placebo for VVA. Vaginal estrogen is superior to lubricants and moisturizers in studies lasting at least 6-12 months. [Ibid., Lethaby, A. et al. Cochrane Database. Syst. Rev. 2016;8:CD001500. doi:10.1002 / 14651858.CD001500.pub3, citing Jokar, A. et al. Intl J. Community Based Nurs. Midwifery (2016) 4:69-78]. Recommended doses are generally two weeks of topical daily application as an offensive therapy, followed by twice-weekly applications as a maintenance therapy. [Ibid., Salwowska, NM, et al. J. Cosmet. Dermatol. (2016) 15:520-26].

[0064] Ospemifene is the only selective estrogen receptor modulator (SERM) indicated for the treatment of VVA. It is approved by the US Food and Drug Administration (FDA) for the treatment of moderate to severe dyspareunia and by the European Medical Agency (EMA) for the treatment of moderate to severe VVA in women with or without a uterus who are not candidates for local estrogen therapy [Ibid., citing De Gregorio, MW, et al. Steroids (2014) 90:82-93]. It exerts a positive effect on the vaginal epithelium and at the same time has neutral or minimal effects on other estrogen-dependent organs. In particular, it exerts a neutral effect on the endometrium and cardiovascular system and, in preclinical studies on the breast, appears to have an anti-estrogenic effect. It is used at a dose of 60 mg per day. The effect of VVA on symptoms is visible after 4 weeks of treatment, such as an increase in superficial cells, a decrease in basal cells, and a decrease in vaginal pH. [Ibid., citing Alvisi, S. et al. Gynecol. Endocrinol. (2017) 33:946-50]. Significant effects on symptoms such as dryness and dyspareunia have been shown to occur after 12 weeks of treatment. [Ibid., citing Goldstein, SR et al. Climacteric. (2014) 17:173-82]. The efficacy of ospemifene at the histological level in both vaginal and vulvar tissues has been demonstrated by observing an increase in the thickness, glycogen content, proliferation index, and vaginal estrogen receptor alpha (ERα) of the vaginal and vulvar epithelium [Ibid., citing Alvisi, S. et al., Gynecol. Endocrinol. (2017) 33:946-50, Alvisi, S. et al. J. Sex. Med. (2018) 15:1776-1784]. Ospemifene has also been shown to improve atrophy of the vulvar vestibule and normalize vestibular sensitivity by increasing the sensory threshold at the vulvar level [Ibid., citing Goldstein, SW et al. Sex. Med. (2018) 6:154-61].Short-term studies have also shown it to increase the ratio of types I and III collagen at the vaginal level, suggesting possible beneficial long-term effects on vaginal connective tissue [ibid. citing Alvisi, S. et al. J. Sex Med. (2018) 15:1776-84].

[0065] Prasterone (dehydroepiandrosterone) has been introduced on the market for the treatment of VVA. It functions as a precursor of the intracellular sex steroids androgens and estrogens. Because the conversion occurs intracellularly, serum estradiol remains within normal values ​​in postmenopausal women, thereby possibly avoiding the risk of systemic effects [Ibid., citing Martel, C. et al. J. Steroid Biochem. Mol. Biol. (2016) 159:142-53]. The efficacy of dehydroepiandrosterone (DHEA) has been demonstrated in a prospective, randomized, double-blind, placebo-controlled Phase III clinical trial that investigated the effect of daily intravaginal prasterone (6.5 mg) on ​​four co-primary objectives: percentage of vaginal parabasal cells, percentage of vaginal surface cells, vaginal pH, and moderate to severe dyspareunia, identified by women as the most bothersome VVA symptom. It may also be effective in reducing libido with possible effects on nerve endings, but more scientific evidence is needed regarding this aspect [ibid., citing Labrie, F. et al. Menopause (2018) 25:1339-53]. The endometrium is not affected by DHEA because the enzymes required to convert DHEA to estrogen are not present in the endometrium. No systemic increase in estrogen levels has been reported, but a history of breast cancer remains a contraindication.

[0066] Energy-based devices may have several potential advantages in treating VVA patients. [Ibid., citing ACOG Position Statement. Fractional Laser Treatment of Vulvovaginal Atrophy and US Food and Drug Administration Clearance. The American College of Obstetricians and Gynecologists; Washington, DC, USA: 2016].

[0067] For example, lasers or radiofrequency waves act by heating the connective tissue of the vaginal wall to 40°C-42°C. In this way, they are said to induce collagen contraction, neocollagenesis, angiogenesis, and growth factor infiltration, ultimately reinvigorating and restoring elasticity and moisture of the vaginal mucosa. The proposed mechanism is the activation of heat shock proteins and tissue growth factors to stimulate new collagen synthesis and epithelial remodeling [ibid. citing Salvatore, S. et al. Curr. Opin. Obstet. Gynecol. (2015) 27:504-8].

[0068] The efficacy of laser therapy in treating VVA has been suggested by improvements in GSM symptoms, VHI scores, and Female Sexual Function Index (FSFI) in many studies, with efficacy at least comparable to that of topical estrogen-based treatments [Ibid., citing Salvatore, S. et al. Climacteric. (2015) 18: 219-225, Gambacciani, M. et al. Climateric. (2018) 21: 148-52]. However, none of these studies were sham or placebo controlled, and the lack of sufficient information, especially regarding long-term safety, led the FDA to issue a warning against indiscriminate marketing of laser treatments in 2018 [Ibid., citing Food and Drug Administration FDA Warns Against Use of Energy-Based Devices to Perform Vaginal'Rejuvenation'or Vaginal Cosmetic Procedures: FDA Safety Communication]. The authors generally suggest that the procedure is well tolerated, quick and painless, although there have been reports of increased vaginal pain, scarring, fibrosis, and vaginal wall lacerations [ibid. citing Gordon, C. et al. Menopause (2019) 26:423-27].

[0069] The radiofrequency devices most commonly used by gynecologists are transcutaneous temperature-controlled radiofrequency (TTCRF) and, more recently, low-energy dynamic quadripolar radiofrequency (DQRF). The mechanism of treatment is to induce anatomical remodeling of the vaginal and vulvar tissues. There are several small studies that attest to its effectiveness on vaginal symptoms, sexual function, and urinary symptoms, but again, they were small and nonrandomized studies [ibid., Caruth, JC. Surg. Technol. Int. (2018) 32:145-49, citing Vicariotto, F. & Raichi, M. Minerva Ginecol. (2016) 68:225-36, Vicariotto, F. et al. Minerva Ginecol. (2017) 69:342-49].

[0070] The use of vaginal dilators to prevent vaginal stenosis due to pelvic radiation is often recommended for gynecologic cancer patients, but data supporting their effectiveness are conflicting [Huffman,LB et al.Gynceol.Oncol.(2016)140(2):359-68, [Miles,T.,Johnson,N.Cochrane Database of Systematic Reviews(2014)9:CD007291, citing Johnson,N.et al.,BJOG(2010)117(5):522-31], and compliance with dilator use is poor. [Ibid., citing Friedman,LC et al.Int.J.Radiat.Oncol.Biol.Phys.(2011)80(3):751-57]. Dilation involves inserting and rotating a penis-shaped device into the vagina for approximately 5 minutes approximately 3 times a week to stretch the skin. On the other hand, dilation may separate adhesions formed by the delaminated epithelium, thus possibly preventing stricture. [Ibid., Faithfull, S. & Wells, M. Supportive Care in Radiotherapy. Edinburgh: Churchill Livingstone (2003); Hassey-Dow, K. Nursing care in Radiation Oncology; Philadelphia: WB Saunders (1992); citing Krumm, SJP psychosomatic Obstetric Gynaecology (1993) 14(1):51-63; Rice A. Intl J. Palliative Nursing (2000) 6(80):392-97; it is also plausible that stretching the vagina during the inflammatory phase of radiation therapy may cause further scarring and promote further damage, both physically and psychologically.

[0071] Treatment Options for Breast Cancer Survivors For women with past or ongoing breast cancer, unfortunately, treatment options are limited. All hormone-based treatments, including vaginal isoflavone-based soy therapy, are contraindicated due to the lack of safety studies in this cohort of women. Non-hormonal approaches are the first choice during or after breast cancer [ibid., citing American College of Obstetricians and Gynecologists' Committee on Gynecologic Practice. Farrell R. ACOG. COMMITTEE OPINION No. 659. The Use of Vaginal Estrogen in Women with a History of Estrogen-Dependent Breast Cancer. Obstet. Gynecol. 2016;127:e93-e96]. Therefore, the options are to offer these women moisturizers and vaginal lubricants, laser or radiofrequency treatments.

[0072] The described invention provides a soothing, less aggressive alternative. Summary of the Invention

[0073] According to one aspect, the present disclosure provides a cosmetic composition formulated for topical application, the cosmetic composition comprising an aqueous gel component comprising hyaluronic acid, a plant raw material component comprising decarboxylated cannabidiol (CBD) representing about 20% of the isolate, and a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid, wherein the composition is a slightly viscous, non-occlusive aqueous liquid, the finished product has a pH in the range of about 4.0 to about 5.0, inclusive, and the composition is not psychoactive.

[0074] In some embodiments of the cosmetic composition, the hyaluronic acid comprises about 0.10 to about 0.50 weight percent (inclusive) low molecular weight hyaluronic acid (LMWHA) and about 0.50 to about 1.50 weight percent (inclusive) high molecular weight hyaluronic acid (HMWHA), with the ratio of HMWHA to MMWHA ranging from 1:0.07 to 1:1. In some embodiments, the molecular weight of the HMWHA ranges from 1,000 to 1,800 kDa (inclusive). In some embodiments, the molecular weight of the HMWHA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa. In some embodiments, the molecular weight of the LMWHA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 1 kDa to less than 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa. In some embodiments, the decarboxylated CBD representing 20% ​​of the isolate is in the form of a THC-free nano-infused water soluble powder. In some embodiments, the cosmetic stabilizing system comprises about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate and about 0.05% to about 0.50% by weight (inclusive) of arginine anisate. In some embodiments, the viscosity of the composition ranges from 5000 to 7500 centipoise (inclusive) at room temperature. In some embodiments, the composition comprises about 1.0% to about 5.0% by weight (inclusive) of a THC-free nano-infused water-soluble powder comprising about 20% decarboxylated CBD.

[0075] According to another aspect, the present disclosure provides a method for promoting and maintaining vaginal-vulvar tissue vitality and vaginal-vulvar tissue health in a female subject in need thereof, comprising topically administering to the subject a cosmetic composition comprising an aqueous gel component comprising hyaluronic acid, a botanical ingredient comprising decarboxylated cannabidiol (CBD) representing 20% ​​w / w of the isolate, and a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid, wherein the composition is a slightly viscous, non-occlusive aqueous liquid, wherein the pH of the composition is in the range of about 4.0 to about 5.0, inclusive, wherein the composition is not psychoactive, wherein the therapeutic effects of the aqueous gel component and the botanical ingredient component may be complementary, and wherein the composition promotes and maintains vaginal-vulvar tissue vitality and tissue health.

[0076] In some embodiments of the method, the female subject in need thereof is a female subject susceptible to or experiencing vaginal-vulvar symptoms of trauma, injury or injury, or a female subject experiencing urogenital symptoms of trauma, injury or injury. In some embodiments of the method, the female subject in need thereof is a menopausal subject, or the female subject in need thereof is a diabetic subject, or the female subject in need thereof is a subject undergoing, undergoing, or undergoing treatment, including radiation therapy, for treating gynecological cancer, or the female subject in need thereof is a breast cancer survivor. In some embodiments, the gynecological cancer is endometrial cancer, cervical cancer, ovarian cancer, or vulvar cancer. In some embodiments, the parameters of vaginal-vulvar tissue vitality include one or more of improved tissue strength, proper vaginal pH, reduced susceptibility to trauma / mechanical injury, reduced inflammation, reduced itch, improved wound healing, and improved tissue elasticity. In some embodiments, the cosmetic composition is effective in restoring damaged tissue to healthy tissue. In some embodiments, the composition regulates vaginal pH, or improves healing and rejuvenation of injured tissue, or reduces susceptibility to trauma or mechanical injury, or reduces symptoms of trauma, injury or injury, or reduces clinical signs of dryness and inadequate hydration (e.g., loss of elasticity, inflammation), or reduces itching, or a combination thereof, compared to an untreated control. In some embodiments, the improvement in healing and rejuvenation of injured tissue comprises improved tissue strength. In some embodiments, the symptoms of trauma, injury or injury comprise one or more of dryness, burning, irritation, discomfort, or pain. In some embodiments, the clinical signs of dryness and inadequate hydration comprise loss of elasticity, inflammation, or both. In some embodiments, the hyaluronic acid comprises about 0.10% to about 0.50% (inclusive) by weight low molecular weight hyaluronic acid (LMWHA) and about 0.50% to about 1.50% (inclusive) by weight high molecular weight hyaluronic acid (HMWHA), with the ratio of HMWHA to LMWHA ranging from 1:0.7 to 1:1.In some embodiments, the molecular weight of the HMWHA ranges from 1,000 to 1,800 kDa, inclusive. In some embodiments, the molecular weight of the HMWHA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa. In some embodiments, the molecular weight of the LMWHA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 10 kDa, at least 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa. In some embodiments, the decarboxylated CBD is in the form of a THC-free nano-infused water-soluble powder. In some embodiments, the cosmetic stabilization system comprises about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate and about 0.05% to about 0.50% by weight (inclusive) of arginine anisate. In some embodiments, the viscosity of the composition ranges from 5000 to 7500 centipoise (inclusive) at room temperature. In some embodiments, the composition comprises about 1.0% to about 5.0% (inclusive) THC-free nano-infused water soluble powder containing about 20% decarboxylated CBD.

[0077] According to another aspect, the disclosure provides a method for promoting and maintaining perianal tissue vitality and perianal tissue health in a subject having hemorrhoidal disease, the method comprising topically administering to the subject a cosmetic composition comprising an aqueous gel component comprising hyaluronic acid, a plant raw material component comprising decarboxylated cannabidiol (CBD) representing 20% ​​w / w of the isolate, and a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid, wherein the composition is a slightly viscous, non-occlusive aqueous liquid, wherein the pH of the composition is in the range of about 4.0 to about 5.0, inclusive, wherein the composition is not psychoactive, wherein the therapeutic effects of the aqueous gel component and the plant raw material component may be complementary, and wherein the composition reduces one or more dermatological symptoms of the hemorrhoidal disease.

[0078] In some embodiments of the method, the hemorrhoidal disease comprises external hemorrhoidal tissue. In some embodiments, the composition improves the healing and rejuvenation of external hemorrhoidal tissue, or reduces the susceptibility of external hemorrhoidal tissue to trauma or mechanical injury, or reduces symptoms of trauma, injury or injury, or reduces clinical signs of dryness and inadequate hydration of external hemorrhoidal tissue, or reduces itching, or a combination thereof, compared to an untreated control. In some embodiments, the improvement in the healing and rejuvenation of external hemorrhoidal tissue comprises improved tissue strength. In some embodiments, the symptoms of trauma, injury or injury comprise one or more of dryness, burning sensation, irritation, discomfort, or pain. In some embodiments, the clinical signs of dryness and inadequate hydration comprise loss of elasticity, inflammation, or both. In some embodiments, the hyaluronic acid comprises about 0.10% to about 0.50% by weight (inclusive) low molecular weight hyaluronic acid (LMWHA) and about 0.50% to about 1.50% by weight (inclusive) high molecular weight hyaluronic acid (HMWHA), with the ratio of HMWHA to LMWHA ranging from 1:0.7 to 1:1. In some embodiments, the molecular weight of the HMWHA ranges from 1,000 to 1,800 kDa (inclusive). In some embodiments, the molecular weight of the HMWHA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa. In some embodiments, the molecular weight of the LMWHA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 10 kDa, at least 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa. In some embodiments, the decarboxylated CBD is in the form of a THC-free nano-infused water soluble powder. In some embodiments, the cosmetic stabilizing system comprises from about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate, and from about 0.05% to about 0.50% by weight (inclusive) of arginine anisate.In some embodiments, the viscosity of the composition ranges from 5000 to 7500 centipoise (inclusive) at room temperature. In some embodiments, the composition comprises about 1.0% to about 5.0% (inclusive) THC-free nano-infused water soluble powder containing about 20% decarboxylated CBD. [Brief description of the drawings]

[0079] [Figure 1] A diagram of the anatomy of the skin is presented. Adapted from Stedman's Medical Dictionary, 27th Ed., Lippincott, Williams & Wilkins, Baltimore, MD (2000), at 1647. [Diagram 2] 1 shows the layers of the epidermis from the stratum corneum to the dermis. [Diagram 3] Shown are micrographs of L929 fibroblast cytotoxicity test: A Complete medium (control); B Complete medium + miniHA™; C Complete medium + HA-Oligo. No morphological cytotoxicity was observed. [Figure 4] FIG. 1 is a plot of relative growth rate (RGR) versus concentration of HA oligosaccharides (%, w / v) added to in vitro cultures of L929 fibroblasts. [Figure 5A] The results of a moisture retention experiment using HA are shown in Fig. 1. A bar graph showing the moisture retention ability of low molecular weight hyaluronic acid miniHA (trademark) (molecular weight 8300 Da) measured by a stratum corneum moisture meter before topical application of the sample and 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours after application is shown. [Figure 5B] The results of a moisture retention experiment using HA are shown in the form of a bar graph showing the moisture retention ability of high molecular weight hyaluronic acid (molecular weight 1,170 kDa) measured with a stratum corneum moisture meter before application of the sample and 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours after application. [Figure 6]Graph of stratum corneum moisture meter value (%) vs. time for 0.1% miniHA, 0.2% miniHA, and 0.5% HA. As shown, the higher concentration of miniHA, i.e., 0.5% miniHA™, has better moisture retention capacity than either 0.1% miniHA or 0.2% miniHA. [Figure 7] 1 is a graph of stratum corneum moisture content (%) versus time for 0.2% HA (molecular weight 1,630,000 DA), 0.2% miniHA™ (molecular weight 8,300), and 0.1% HA + 0.1% miniHA. As shown, when miniHA™ was used in combination with HMWHA, the moisturizing effect was superior to either alone. [Figure 8A] The results of the moisture retention test are shown. Graph of stratum corneum moisture meter value (%) vs. time for 0.1% miniHA, 0.1% HA-1630kDa, and 0.1% HA-270kDa. As shown, the lower the molecular weight of the HA, the better the moisture retention. The MiniHA group had the highest values ​​of skin hydration at each time point. [Figure 8B] TEWL test results. Transepidermal water loss (TEWL) versus time graph for 0.1% miniHA, 0.1% HA-1630kDa, and 0.1% HA-270kDa. The data shows that the higher the molecular weight of HA, the greater the reduction in skin moisture. [Figure 9A] The results of the moisture retention test are shown in the form of a bar graph of the stratum corneum moisture meter value (%) versus time for 0.1% miniHA+0.1% HA-270 kDa. [Figure 9B] The results of the TEWL study are shown. A bar graph of transepidermal water loss (TEWL) versus time for 0.1% miniHA + 0.1% HA-270kDa is shown. Combined, the moisturizing effect is superior to either ingredient alone. [Figure 10] FIG. 1 is a cross-section through human reconstructed epidermis. [Figure 11] FIG. 1C is a cross-section through a human reconstructed full-thickness sin. [Figure 12] Schematic of the experimental system: The surface applied product penetrates the remodeled human tissue and a certain amount of the product is retained by the tissue structure. [Figure 13] Graph of % absorption versus time for reconstructed epidermis, full thickness reconstructed skin, and dermis. Dermal results were mathematically calculated by subtracting full thickness and epidermal absorption data. [Figure 14] 1 is a microscopic image of human heratinocytes (HaCaT line) in culture used in the study of Example 4. [Figure 15] 1 is a bar graph showing relative cell viability (OD 550 nm) versus RS-0198A at concentrations of 3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003%, and 0.001% compared to untreated control. **** denotes statistical significance at p-value <0.0001. * denotes statistical significance at p-value <0.05. [Figure 16] 1 is a bar graph showing relative cell viability (OD550 nm) versus RS-0198B at concentrations of 3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003%, and 0.001% compared to untreated control. **** denotes statistical significance at p-value <0.0001. ** denotes statistical significance at p-value <0.01. [Figure 17] 1 is a bar graph showing relative cell viability (OD550nm) versus RS012221 at concentrations of 3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003%, and 0.001% compared to untreated control. **** denotes statistical significance at p-value <0.0001. [Figure 18] 1 is a bar graph showing wound area after 24 hours of treatment of human keratinocytes (HaCaT) with 0.00001%, 0.0001%, 0.001% RS-0198A, and 0.0001%, 0.001%, and 0.01% RS-0198B compared to untreated controls. 20ng / ml human EGF was included as a positive control. * indicates statistical significance at p-value <0.05. ** indicates statistical significance at p-value <0.01. [Figure 19]1 is a bar graph showing wound area after 24 hours of treatment of human keratinocytes (HaCaT) with RS012221 at concentrations of 0.001% and 0.01% versus untreated control. Human EGF at 20ng / ml was included as a positive control. ** denotes statistical significance at p-value <0.01. **** denotes statistical significance at p-value <0.0001. [Figure 20] 1 is a bar graph showing wound healing after 24 hours of treatment of human keratinocytes (HaCaT) with 0.00001%, 0.0001%, and 0.001% RS-0198A and 0.0001%, 0.0001%, and 0.01% RS-0198B compared to untreated controls. 20ng / ml human EGF was included as a positive control. * indicates statistical significance at p-value <0.05. ** indicates statistical significance at p-value <0.01. [Figure 21] 1 is a bar graph showing wound healing after 24 hours of treatment of human keratinocytes (HaCaT) with RS012221 at concentrations of 0.001% and 0.01% compared to untreated controls. Human EGF at 20ng / ml was included as a positive control. ** denotes statistical significance at p-value <0.01. **** denotes statistical significance at p-value <0.0001. [Figure 22A] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. 20 ng / ml human EGF was included as a positive control. Control, t=0 hours. [Figure 22B] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 h) and 24 h after treatment. 20 ng / ml human EGF was included as a positive control. Control, t=24 h. [Figure 22C] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. EGF t=0 hours. [Figure 22D]Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. EGH t=24 hours. [Figure 22E] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.00001%, t=0 hours. [Figure 22F] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.00001%, t=24 hours. [Figure 22G] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.0001%, t=0 hours. [Fig. 22H] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.0001%, t=24 hours. [Figure 22I] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.001%, t=0 hours. [Figure 22J] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198A, 0.001%, t=24 hours. [Figure 22K]Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.0001%, t=0 hours. [Figure 22L] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.0001%, t=24 hours. [Figure 22M] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.001%, t=0 hours. [Figure 22N] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.001%, t=24 hours. [Figure 22O] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.01%; t=0 hours. [Figure 22P] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS-0198B, 0.01%, T=24 hours. [Figure 23A] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 h) and 24 h after treatment. Human EGF at 20 ng / ml was included as a positive control. Control, 0.5%, t=0 h. [Figure 23B]Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 h) and 24 h after treatment. Human EGF at 20 ng / ml was included as a positive control. Control, 0.5%, t=24 h. [Figure 23C] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 h) and 24 h after treatment. Human EGF at 20 ng / ml was included as a positive control. EGF, 20 ng / ml, t=0 h. [Figure 23D] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 h) and 24 h after treatment. Human EGF at 20 ng / ml was included as a positive control. EGF, 20 ng / ml, t=24 h. [Figure 23E] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RSO12221, 0.01%, t=0 hours. [Figure 23F] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS12221, 0.01%, t=24 hours. [Figure 23G] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RS012221, 0.001, t=0 hours. [Figure 23H] Shown are microscopic images of wound healing from a scratch performed on a human keratinocyte (HaCaT) monolayer immediately before treatment (0 hours) and 24 hours after treatment. Human EGF at 20 ng / ml was included as a positive control. RSO12221, 0.001%, t=24 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0081] As used herein, the term "about" means ±20% of the numerical value of the number with which it is used, so about 50% means a range of 40% to 60%.

[0082] As used herein, the term "active agent" refers to an ingredient, component or constituent of the composition that is responsible for the intended cosmetic benefit.

[0083] As used herein, the term "administer" means to give or apply. As used herein, the term "administer" includes in vivo administration as well as direct administration to tissue ex vivo.

[0084] The term "analog" as used herein refers to a compound whose structure is related to the structure of another compound, but whose chemical and biological properties may differ significantly. A "direct analog" has chemical and pharmacological similarity to an existing compound, whereas a "structural analog" has only structural similarity and may differ in one or more atoms, functional groups, or substructures, and has different physical, chemical, biochemical, or pharmacological properties. The term "functional analog" as used herein refers to a chemically distinct compound that exhibits similar pharmacological properties.

[0085] The term "angiogenesis", as used herein, refers to the organization of new blood vessels from pre-existing functional vessels. Under physiological conditions, angiogenesis depends on the balance of positive and negative angiogenic regulators within the vascular microenvironment and requires the functional activity of several molecules, including angiogenic factors, extracellular matrix proteins, adhesion receptors, and proteolytic enzymes. Normally, endothelial cell turnover is very low due to the balance of stimulators and inhibitors. However, under certain conditions (e.g., low pO2, low pH, hypoglycemia, mechanical stress, injury, immune / inflammatory stimuli, tumors), endothelial proliferation increases dramatically, a phenomenon called activation of the "angiogenic switch". Under these circumstances, the influence of activators outweighs that of inhibitors. These regulators of angiogenesis include growth factors, proteases and protease inhibitors, cytokines and chemokines, as well as a miscellaneous catch-all category termed "intrinsic regulators". Examples of regulators of angiogenesis are listed in Table 1 [Gerritsen, ME. Chapter 8-Angiogenesis, In Handbook of Physiology, Microcirculation, 2d Ed. Elsevier, Inc. (2008), Tuma, RF, et al Eds, pp. 351-83]. [Table 1]

[0086] Hypoxia is one of the major drivers of angiogenesis. Hypoxia-inducible factor 1 (HIF) is a heterodimer of two DNA-binding proteins, HIF-1α and HIF-1β (also known as aryl hydrocarbon nuclear translocator, ARNT). Under normal oxygen tension, HIF-1α undergoes post-translational modification (by prolyl hydroxylases), leading to recognition of HIF-1α by ubiquitin ligases (Hippel-Lindau proteins). Ubiquitinated HIF-1α is then targeted for destruction by the proteasome. In contrast, during hypoxia, the activity of prolyl hydroxylases is reduced, stabilizing HIF-1α and allowing it to form heterodimers with HIF-1β. HIF heterodimers bind to specific hypoxia response elements, thereby leading to upregulation of the expression of many hypoxia-inducible genes (e.g., adrenomedullin, angiopoietin-2, cyclooxygenase-2, endothelin-1, endothelin-2, hepatocyte growth factor, interleukin-8, leptin, migration inhibitory factor, monocyte chemotactic protein-1, nitric oxide synthase, placentacle growth factor, plasminogen activator inhibitor-1, stanniocalcin 1, stromal cell-derived factor 1 (CXCL12), TGFα, TGF-β1, TGF-β3, Tie-1, urokinase receptor, VEGF-A, VEGFR-1), many of which are known to play important roles in angiogenesis. One of the most important genes upregulated by hypoxia-inducible factor 1 (HIF) is VEGF-A. When hypoxia sets up a VEGFA source, endothelial cells migrate towards the gradient of VEGFA that is sensed by the VEGFR2 receptor. Iruela-Arispe, M. and Zovein, A. In Fetal and Neonatal Physiology 5 th Ed.(2017)Elsevier,Inc.)Vol.1:85-89.e2,

[0087] The term "anorectal" as used herein refers to both the anus and the rectum. The terms "anus" or "anal orifice" as used herein refer to the lower opening of the digestive tract located in the cleft between the buttocks through which feces is extruded. The term "rectum" as used herein means relating to the rectum and refers to the terminal portion of the digestive tract extending from the rectosigmoid junction to the anal canal. The term "anal canal" as used herein refers to the most distal portion of the lower GI tract / large intestine between the anal verge of the lower perineum (anal orifice, anus) and the upper rectum.

[0088] The term "anti-inflammatory," as used herein, refers to reducing inflammation (redness, swelling, and pain) in the body by inhibiting inflammatory mediators in the body that cause inflammation.

[0089] The term "anti-irritant" as used herein refers to preventing or reducing soreness, roughness, or inflammation on a body part.

[0090] The term "apoptosis" or "programmed cell death" refers to a highly regulated and active process that contributes to biological homeostasis consisting of a series of biochemical events that result in a variety of morphological changes, including blebbing, changes to the cell membrane, e.g., loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and fragmentation of chromosomal DNA, without causing damage to the organism.

[0091] Apoptotic cell death is induced by many different factors and involves many signaling pathways, some dependent on caspase proteases (a class of cysteine ​​proteases) and some independent of caspase. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial responses to stress, and cytotoxic T cells, which lead to the activation of apoptotic signaling pathways.

[0092] Caspases involved in apoptosis transmit the apoptotic signal in a proteolytic cascade in which caspases cleave and activate other caspases, which then degrade other cellular targets causing cell death. Caspases at the top end of the cascade include caspase 8 and caspase 9. Caspase 8 is the first caspase engaged in response to death domain (DD)-containing receptors such as Fas.

[0093] Receptors of the TNF receptor family are involved in the induction of apoptosis and inflammatory signaling. The Fas receptor (CD95) mediates apoptosis signaling by Fas ligand expressed on the surface of other cells. Fas-FasL interaction plays an important role in the immune system, and defects in this system lead to autoimmunity, and Fas-mediated apoptosis has been shown to eliminate autoreactive lymphocytes. Fas signaling is also involved in immune surveillance to eliminate transformed and virus-infected cells. Binding of Fas to oligomerized FasL on another cell activates apoptosis signaling through a cytoplasmic domain called the death domain (DD), which interacts with signaling adaptors including FAF, FADD, and DAX to activate the caspase proteolytic cascade. Caspase 8 and caspase 10 are activated first, which then cleave and activate various cellular substrates leading to downstream caspases and cell death.

[0094] Mitochondria participate in apoptosis signaling pathways through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase 9 and the rest of the caspase pathway. Smac / DIABLO is released from mitochondria and inhibits IAP proteins that normally interact with caspase 9 to inhibit apoptosis. Regulation of apoptosis by Bcl-2 family proteins occurs when family members form a complex that enters the mitochondrial membrane, thereby regulating the release of cytochrome c and other proteins. TNF family receptors that trigger apoptosis directly activate the caspase cascade, but can also activate Bid, a Bcl-2 family member that activates mitochondria-mediated apoptosis. Another Bcl-2 family member, Bax, is activated by this pathway to localize and permeabilize the mitochondrial membrane, which allows the release of cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation and block apoptosis. Similar to cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria, from which it is released upon apoptotic stimuli. While cytochrome C is involved in caspase-dependent apoptosis signaling, AIF release stimulates caspase-independent apoptosis, translocates to the nucleus, and binds to DNA. DNA binding by AIF stimulates chromatin condensation and DNA fragmentation, possibly through recruitment of nucleases.

[0095] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, which leads to the autocleavage and activation of caspase 9. Caspases 3, 6, and 7 are downstream caspases that are activated by upstream proteases and act to cleave cellular targets.

[0096] Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, leading to the formation of transmembrane pores that trigger apoptosis, presumably via caspase cleavage, although a caspase-independent mechanism of granzyme B-mediated apoptosis has been suggested.

[0097] Fragmentation of the nuclear genome by multiple nucleases that are activated by apoptotic signaling pathways to create nucleosomal ladders is a cellular response characteristic of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD). DFF / CAD is activated during apoptosis by cleavage of its associated inhibitor ICAD by caspase proteases. DFF / CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and likely recruit CAD to chromatin. Another apoptosis-activating protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but localizes to mitochondria in normal cells. EndoG may play a role in mitochondrial genome replication and apoptosis. Apoptotic signaling causes the release of EndoG from mitochondria. EndoG and DFF / CAD pathways are independent, as the EndoG pathway still occurs in cells that lack DFF.

[0098] Hypoxia and hypoxia followed by hypoxia can cause cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3), a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or enhance apoptosis by many stimuli that activate the mitochondrial cell death pathway. Loberg, RD, et al., J. Biol. Chem. 277(44):41667-673 (2002). It has been demonstrated to induce activation of caspase 3 and activate the proapoptotic tumor suppressor gene p53. It has also been suggested that GSK-3 promotes the activation and translocation of Bax, a proapoptotic Bcl-2 family member that upon aggregation and mitochondrial localization induces cytochrome c release. Akt is a key regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of the antiapoptotic effects of Akt.

[0099] The term "apply" as used herein refers to placing on or laying on in contact with and spreading over.

[0100] As used herein, the phrase "arginine compound" is used to refer to arginine, its salts, conjugates, or analogs thereof.

[0101] The term "arginine" is used herein to describe a molecule or compound that contains one amino group, one guanidino group, and one carboxylic group. Arginine is a solid and a known skin and eye irritant. At physiological pH, the carboxylic acid is deprotonated (-COO - ), and the amino group is protonated (-NH3 + ), the guanidino group is also protonated to form the guanidinium (-C-(NH2) 2+ ), making arginine a positively charged aliphatic amino acid.

[0102] Arginine has the molecular formula CH 15N4O2 and is generally shown as formula I. [ka]

[0103] In some embodiments, the arginine compound can be in one or more isomeric forms represented by Formulas II and III below. [ka]

[0104] L-arginine, the enantiomer of D-arginine, is also known as L(+)-arginine, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, (2S)-2-amino-5-guanidinopentanoic acid, (S)-2-amino-5-guanidinopentanoic acid, and (S)-2-amino-5-guanidinovaleric acid. It is considered the physiologically active isomer of arginine. It plays a role in several essential biochemical processes. L-arginine is the conjugate base of L-arginium(1+) (i.e., it has one less H atom and one more negative charge than the acid that formed it) and the conjugate acid of L-arginate (i.e., it has one more H atom and one more positive charge than the base that formed it).

[0105] D-arginine, the enantiomer of L-arginine, is also known as D-2-amino-5-guanidinovaleric acid, (2R)-2-amino-5-guanidinopentanoic acid. It was thought that D-arginine may have slightly different properties than L-arginine (discussed above). D-arginine is slightly soluble in water and is considered a moderately acidic compound based on its pKa. D-arginine is the conjugate base of D-arginium(1+) and the conjugate acid of D-arginic acid.

[0106] In some embodiments, the arginine compound may be in one or more conjugated forms represented by Formulas IV and V below. [ka]

[0107] In some embodiments, the arginine compound can be in the form of one or more analogs represented by formula VI: [ka] During the ceremony, R1 is, represents a hydrogen atom, a hydroxyl group, an acyl or acyloxy radical, or an amino acid substituted or unsubstituted on its free α-amino function, linked by a peptide bond, R2 is represents a hydroxyl group, an amine, an alkylamine or an alkoxy radical, a silyloxy group, or an amino acid, substituted or unsubstituted on its free α-carboxylic acid function, linked by a peptide bond, and n represents 3 or 4.

[0108] In some embodiments, the arginine compound is obtained from a commercial source, including, but not limited to, VladaChem, Tractus, Phion Ltd, MuseChem, MolPort, Sigma-Aldrich, LGC Standards, AN PharmaTech, ChemFaces, CAPOT, eNovation Chemicals, abcr GmbH, Tyger Scientific, Hairui Chemical, BLD Pharm, Biosynth, Aurum Pharmatech LLC, ApexBio Technology, ChemShuttle, Oakwood Products, DC Chemicals, AA BLOCKS, Yuhao Chemical, Norris Pharm, Tocris Bioscience, R&D Chemicals, Assembly Blocks Pvt.Ltd, Parchem, Angene Chemical, MedChemexpress MCE, Achemo Scientific Limited, Apexmol, Key Organics / BIONET, etc. In some embodiments, the arginine compound is synthesized.

[0109] The term "plant raw material" as used herein refers to fresh or processed (e.g., washed, frozen, dried, sliced, or liquefied) parts of a single species of plant or fresh or processed algae or macroscopic fungi. The term "plant raw material" as used herein refers to ingredients derived from plant raw materials. The term "plant product" refers to a finished labeled product containing plant matter, which may include plant material, algae, macroscopic fungi, or combinations thereof. Depending in part on its intended use, the plant product may be a food, drug, or cosmetic. The term "plant extract" as used herein refers to a product prepared by separating medicamentously active parts of a plant from inactive or inert components by chemical or physical processes. Plant extracts prepared according to some embodiments of the invention described may be obtained by solvents, optionally under pressure and / or heat.

[0110] The term "carrier" as used herein describes a material that does not cause significant irritation to an organism and does not inhibit the biological activity and properties of the active compounds of the compositions of the present invention described. The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal being treated. The carrier may be inert or may have pharmaceutical benefits, cosmetic benefits, or both. The terms "excipient", "carrier", or "vehicle" are used interchangeably to refer to carrier materials suitable for formulation and administration of the pharma-ceutically acceptable compositions described herein. Carriers and vehicles useful herein include any such materials known in the art that are non-toxic and do not interact with other components.

[0111] The term "chemoattractant," as used herein, refers to a substance that attracts specific types of motile cells.

[0112] The term "chemokine" as used herein refers to a family of chemoattractant cytokines that play a key role in leukocyte migration. They are classified into four main subfamilies. CXC, CC, CX3C, and XC all exert their biological effects by interacting with G protein-coupled receptors known as chemokine receptors. Chemokines act through a concentration gradient that mediates migration of cells to areas of high chemokine concentration. In addition to their role in immune system communication, chemokines are also expressed by other cell types. For example, in the CNS, neurons constitutively express CX3CL1 (also known as fractalkine) to control microglial activation via the CX3CL1 / CX3CR1 axis. Chemokine expression is induced after ischemia by the action of inflammatory cytokines such as IL-1, TNFα, and IL-6 on resident and infiltrating cells of the ischemic tissue. Two major chemokine networks are thought to recruit neutrophils and monocytes to ischemic tissues: the CXCL8 (IL-8) / CXCR2 and CCL2 / CCR2 axes. In addition, the CX3CL1 / CX3CR1 axis and SDF-1 (CXCL12) also play important roles in ischemic outcome and neurorepair. The chemokine receptor CXCR4 is the receptor for stromal cell-derived factor-1, SDF-1.

[0113] The term "conjugate acid-base pair" as used herein refers to a proton donor and its corresponding deprotonated species, e.g., benzoic acid (donor) and benzoate (acceptor). The term "conjugate acid" as used herein refers to the acid member of a pair of compounds that differ from each other by gaining or losing a proton. The conjugate acid can release or donate a proton. The term "conjugate base" as used herein refers to the species remaining after the acid has donated its proton, and the conjugate base can accept a proton.

[0114] The term "combination" as used herein refers to an assembly of separate parts or qualities. The term "combining" as used herein refers to placing or adding together.

[0115] The term "complementary" as used herein refers to combining in such a way as to enhance or accentuate the properties of each other.

[0116] The term "complex" as used herein refers to a molecular entity formed by loose association involving two or more constituent molecular entities. The bonds between the components are usually weaker than covalent bonds. The strength of the complex derives from the delocalization and sharing of charges. Coordination complexes, also called coordination compounds (coordination complexes means compounds or ions having a central, usually metal atom or ion, bound by coordinate bonds with a certain number of surrounding ions, groups, or molecules), may or may not be covalently bonded.

[0117] As used herein, the term "ingredient" refers to a component, element, or ingredient.

[0118] The term "composition," as used herein, refers to a mixture of ingredients.

[0119] As used herein, the term "contact" and its various grammatical forms refer to the state or condition of being in contact or in immediate or local proximity.

[0120] The term "controlled release" is intended to refer to any active agent-containing formulation in which the manner and profile of release of the active agent from the formulation is regulated. It refers to immediate and non-immediate release formulations, which include, but are not limited to, sustained release and delayed release formulations. The term "delayed release" is used herein in its conventional sense to refer to a formulation in which there is a time delay between administration of the formulation and the release of the active agent therefrom. "Delayed release" may or may not involve a gradual release of the active agent over an extended period of time, and therefore may or may not be a "sustained release".

[0121] As used herein, the term "cosmetic" refers to articles (other than soap) intended to be rubbed, poured, sprinkled, sprayed, introduced, or otherwise applied to the human body or any part thereof for the purpose of cleaning, beautifying, promoting attractiveness, or altering appearance, and articles intended for use as components of such articles.

[0122] The term "cosmetic composition" as used herein refers to a composition intended to be rubbed, poured, sprinkled, or sprayed, introduced, or otherwise applied to an object or any part thereof for cleaning, beautifying, promoting attractiveness, or altering the appearance, or an article intended for use as a component of such an article, but such term does not include soap.

[0123] As used herein, the term "cosmetic benefit" refers to the result of applying a cosmetic product to the skin with the intent of improving or beautifying its appearance.

[0124] The term "cosmetically acceptable carrier" as used herein refers to a substantially non-toxic carrier that can be used for cosmetic administration, in which the active compound remains stable and is bioavailable. The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal being treated. In addition, it must maintain the stability and bioavailability of the active agent. A cosmetically acceptable carrier is selected with the proposed method of administration in mind, to provide the desired bulk, consistency, etc., when combined with the active agent and other ingredients of a given composition.

[0125] As used herein, the term "cytokine" refers to small soluble protein substances secreted by cells that have various effects on other cells. Cytokines mediate many important physiological functions, including proliferation, development, wound healing, and immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows them to initiate distinct signaling cascades within the cell that ultimately result in biochemical and phenotypic changes in the target cell. Cytokines can act both locally and remotely from the site of release. They include type I cytokines, which encompass many interleukins, and several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules, including TNFα and lymphotoxin; immunoglobulin superfamily members, including interleukin-1 ("IL-1"), and chemokines, a family of molecules that play important roles in a wide variety of immune and inflammatory functions. The same cytokine may affect cells differently depending on the state of the cell. Cytokines often regulate the expression of other cytokines and trigger cascades of other cytokines. Non-limiting examples of cytokines include, for example, IL-1α, IL-β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23 P40, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, TGF-β, IFN-γ, GM-CSF, Gro-α, MCP-1 and TNF-α.

[0126] As used herein, the term "dentate line" refers to the anatomical landmark that divides the anal canal from the lower one-third to the upper two-thirds.

[0127] The term "derivative" as used herein means a compound that can be produced from another compound of similar structure in one or more steps. A "derivative" or "derivative" of a compound retains at least some of the desired functionality of the compound. Thus, an alternative term for "derivative" can be "functional derivative". Derivatives include chemical modifications of a compound, such as akylating, acylating, carbamylating, iodinating, or any modification that derivatizes a compound. Such derivatized molecules include, for example, molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formal groups. Free carboxyl groups can be derivatized to form salts, esters, amides, or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives.

[0128] The term "dyspareunia," as used herein, refers to pain during sexual intercourse.

[0129] As used herein, the terms "emollient" or "moisturizer" are used interchangeably to refer to a complex mixture of chemicals specifically designed to make the outer layer of the skin (epidermis) softer and more supple. Emollients increase skin hydration (water content) by reducing evaporation.

[0130] The term "extracellular matrix" or "ECM" as used herein refers to a scaffold in the external environment of cells with which cells interact through specific cell surface receptors. The extracellular matrix serves many functions, including but not limited to supporting and anchoring cells, separating one tissue from another, and regulating intracellular communication. The extracellular matrix is ​​composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). Examples of fibrous proteins found in the extracellular matrix include collagen, elastin, fibronectin, and laminin. Examples of GAGs found in the extracellular matrix include proteoglycans (e.g., heparin sulfate), chondroitin sulfate, keratan sulfate, and non-proteoglycan polysaccharides (e.g., hyaluronic acid). The term "proteoglycan" refers to a group of glycoproteins that contain a core protein to which one or more glycosaminoglycans are attached.

[0131] The terms "formulation" and "composition" are used interchangeably herein to refer to the described product of the invention, including all active and inactive ingredients.

[0132] As used herein, the term "finished product" refers to a cosmetic composition that has gone through all stages of manufacture, including packaging in its final container.

[0133] The terms "formulation" and "composition" are used interchangeably herein to refer to the described product of the invention, including all active and inactive ingredients.

[0134] As used herein, the term "cure" and its various grammatical forms means to restore to a state of health, well-being, or health.

[0135] As used herein, the term "hydrophilic" refers to a material or substance that has an affinity for polar substances such as water. As used herein, the term "hydrophobic" refers to a material or substance that has an affinity for non-polar or neutral substances.

[0136] As used herein, the term "improve" and its various grammatical forms refer to making into a more desirable or superior state.

[0137] The term "inflammation" as used herein refers to the physiological process by which vascularized tissue responds to injury. See, for example, FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein. During the inflammatory process, cells involved in detoxification and repair are recruited to sites compromised by inflammatory mediators. Inflammation is often characterized by intense infiltration of leukocytes, particularly neutrophils (polymorphonuclear cells), at the site of inflammation. These cells promote tissue damage by releasing toxic substances into the vessel wall or into intact tissue. Traditionally, inflammation has been divided into acute and chronic responses.

[0138] As used herein, the term "acute inflammation" refers to a rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by the accumulation of fluid, plasma proteins, and neutrophil leukocytes. Examples of harmful agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), exogenous foreign bodies (e.g., asbestos) or endogenous (e.g., uric acid crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., corrosive) agents.

[0139] The term "chronic inflammation" as used herein refers to inflammation that is longer term and has an unclear, indefinite termination. Chronic inflammation takes over when acute inflammation persists due to incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring at the same location. Chronic inflammation includes the influx of lymphocytes and macrophages, as well as the proliferation of fibroblasts, which can lead to tissue scarring at the site of prolonged or repeated inflammatory activity.

[0140] The term "immune system" as used herein refers to a complex network of cells, tissues, organs, and the substances they make that help the body fight infections and other diseases. The immune system includes white blood cells and lymphatic organs and tissues, such as the thymus, spleen, tonsils, lymph nodes, lymphatic vessels, and bone marrow. Responses in the immune system can be generally divided into two arms, termed "innate immunity" and "adaptive immunity." The innate arm of the immune system is a non-specific, fast response to pathogens that is primarily involved in the initial inflammatory response, via a number of soluble factors, including the complement system and the chemokine / cytokine system, and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs). The adaptive immune arm includes specific, delayed, long-term responses by various types of cells that create long-term immune memory against specific antigens. It can be further subdivided into cellular and humoral branches, the former mediated primarily by T cells and the latter by B cells.

[0141] The terms "immunomodulation," "immunomodulator," "immunomodulatory," and "immunomodulatory" are used interchangeably herein to refer to substances, agents, or cells that can directly or indirectly enhance or decrease the immune response, for example, by inducing the expression of chemokines, cytokines, and other mediators of the immune response.

[0142] As used herein, the term "immunostimulatory amount" refers to an amount of an immunogenic composition that stimulates an immune response by a measurable amount, as measured, for example, by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay), and major histocompatibility complex (MHC) tetramer assay.

[0143] As used herein, the term "immunosuppressive amount" refers to an amount of an immunosuppressive composition that suppresses an immune response as measured, for example, by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay), and major histocompatibility complex (MHC) tetramer assay.

[0144] As used herein, the term "inflammasome" refers to an inflammatory protein complex that forms following stimulation of intracellular NOD-like receptors. The production of active caspases in the complex processes cytokine proteins into active cytokines.

[0145] The term "lesion" as used herein refers to a traumatic injury or wound.

[0146] The term "injury" as used herein refers to injury, attack, or trauma.

[0147] The term "interleukin (IL)" as used herein refers to a cytokine from a class of homologously related proteins that were first observed to be secreted by and act on white blood cells. Since then, interleukins have been found to be produced by a wide variety of somatic cells. Interleukins regulate cell growth, differentiation, and motility and stimulate immune responses, such as inflammation. Examples of interleukins include interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), and interleukin-17 (IL-17). Interleukin-1-α (IL-1A) and interleukin-1β (IL-1B) are equally potent inflammatory cytokines that activate inflammatory processes [DiPaolo, NC and Shayakhmetov, DM, Nat. Immunol. (2016) 17(8):906-13]. Interleukin 6 (IL-6), produced rapidly and transiently in response to infection and tissue injury, contributes to host defense through stimulation of acute phase responses, hematopoiesis, and immune responses. Its expression is tightly controlled by transcriptional and post-transcriptional mechanisms, but dysregulated continuous synthesis of IL-6 plays pathological effects in chronic inflammation and autoimmunity [Tanaka, T. et al. Cold Spring Harb. Perspect. Biol. (2014) 6(10): a016295]. Interleukin 8 (IL-8) is a proinflammatory cytokine with proangiogenic, pro-proliferative, and anti-inflammatory activities [Kondo, S. et al. J. Invest. Dermatol. (1993) 101(5): 690-4].

[0148] The term "isolated" as used herein refers to an element or compound that has been separated in substantially pure form from the substances with which it may be associated in a biological system or during synthesis. As used herein, the term "substantially pure" refers to a purity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as determined by analytical protocols.

[0149] The term "keratinocyte-derived chemokine (KC / CXCL1) is one of the major attractants of neutrophils in mice and is the functional mouse homolog of human CXCL1 / Gro-α and CXCL8 / IL-8. It binds to the chemokine receptor CXCR2 present on neutrophils. [See Chintakuntlawar, AV & Chodosh, JJ Interferon Cytokine Res. (2009) 29(10):657-666].

[0150] The term "lymphocyte" refers to small white blood cells (leukocytes) that form in lymphoid tissues throughout the organism and that, in normal adults, constitute about 22-28% of the total white blood cell count in the circulating blood, playing a major role in defending the organism against disease. Individual lymphocytes are specialized to commit to respond to a limited set of structurally related antigens through recombination of their genetic material. This commitment, which exists before the first contact of the immune system with a given antigen, is represented by the presence of receptors specific for the antigen's determinants (epitopes) on the lymphocyte's surface membrane. Each lymphocyte has a unique collection of receptors, all of which have identical binding sites. No one set or clone of lymphocytes is the same in the epitopes it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors but also in their functions (ibid.). Two broad classes of lymphocytes are recognized. B lymphocytes (B cells) are the precursors of antibody-secreting cells. T lymphocytes or T cells mediate a wide range of immune functions. These include the ability to help B cells develop into antibody-producing cells, to increase the bactericidal activity of monocytes / macrophages, inhibition of certain immune responses, direct killing of target cells, and recruitment of inflammatory responses. These effects depend on T cell expression of specific cell surface molecules and secretion of cytokines (Paul, WE, "Chapter 1: The immune system: an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippincott-Raven Publishers, Philadelphia, (1999)).

[0151] The term "maintain" as used herein refers to keeping, holding, supporting, preserving, or continuing in a healthy state.

[0152] The term "matrix metalloproteinase (MMP)" as used herein refers to zinc-dependent endopeptidases that can degrade extracellular matrix molecules. The dynamic equilibrium between matrix metalloproteinases and their inhibitors is a key determinant of matrix remodeling (R. Visse and H. Nagase, "Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry," Circulation Research, vol. 92, no. 8, pp. 827-839, 2003). These enzymes are called metalloproteinases because they require zinc or calcium atoms to function properly. Matrix metalloproteinases are involved in wound healing, angiogenesis, and tumor cell metastasis. For example, MMP-12 is a key regulator of macrophage infiltration and inflammation, contributing to retinal vascular dysfunction and pathological angiogenesis.

[0153] The term "macrophage inflammatory protein-1 alpha (MI-1α)" as used herein refers to a chemically inducible chemokine secreted by macrophages. It serves a variety of biological functions, such as recruitment of inflammatory cells, wound healing, inhibition of stem cells, and maintenance of effector immune responses.

[0154] As used herein, the term "microbiome" refers to a distinctive microbial community occupying a rational, well-defined habitat with distinct physicochemical properties that forms a dynamic and interactive micro-ecosystem that is subject to change over time and scale, and is integrated into a macro-ecosystem that includes a eukaryotic host. [Berg, G. et al. Microbiome (2020) 8: 103].

[0155] As used herein, the term "microbiota" includes all living members that form the microbiome.

[0156] As used herein, the term "modulate" means to adjust, alter, adapt, or adjust a particular measure or proportion.

[0157] As used herein, the term "monocyte chemoattractant protein 1 (MCCP-1 / CCL2)" refers to a key chemokine that regulates monocyte / macrophage migration and infiltration.

[0158] The term "MTT assay" as used herein refers to a colorimetric reaction that can be easily measured from cell monolayers plated in 35 mm dishes or multi-well plates. It is widely used to assess cell viability and relies on the enzymatic reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan, as catalyzed by mitochondrial succinate dehydrogenase. Thus, the MTT assay relies on mitochondrial respiration and serves to indirectly assess the cellular energy capacity of cells.

[0159] The term "neutrophilic granulocytes" or polymorphonuclear neutrophils (PMNs) as used herein refers to the most abundant white blood cells in humans and mice. They are characterized by the multilobed shape of their nuclei. Neutrophils are the first white blood cells recruited to sites of acute inflammation in response to chemotactic cues such as CXCL8 (interleukin-8, IL-8) produced by stressed tissue cells and tissue-resident immune cells such as macrophages. Neutrophils exhibit a range of biological functions important for both innate and adaptive immune responses. Neutrophils can produce many cytokines and chemokines upon stimulation and in this way can interact with endothelial cells, dendritic cells, macrophages, natural killer cells, T lymphocytes, and B lymphocytes. Through all these interactions, neutrophils can activate or downregulate both innate and adaptive immunity. [See Rosales, C. et al. J. Immunol. Res. (2017):9748345].

[0160] As used herein, the term "normal healthy control subject" refers to a subject that has no symptoms or other clinical evidence of vaginal / vulvar or genitourinary trauma, injury or injury.

[0161] The term "nuclear factor kappa B (NF-kappa B)" as used herein refers to a family of inducible transcription factors that regulate cytokine and chemokine signaling. This family switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and inhibitors of apoptosis, resulting in the amplification of the inflammatory response [Barnes, PJ, (2016) Pharmacol. Rev. 68: 788-815]. The molecular pathway involved in the activation of NF-kappa B involved several kinases. The classical (canonical) pathway for inflammatory stimuli and infection to activate NF-kappa B signaling involves the IKK (inhibitor of kappa B kinase) complex, which consists of two catalytic subunits, IKK-α and IKK-β, and the regulatory subunit IKK-γ (or NFkappa B essential modulator [ibid., Hayden, MS and Ghosh, S (2012) Genes Dev. 26:203-234]. The IKK complex phosphorylates NF-κB-bound IκBs and targets them for degradation by the proteasome, thereby releasing NF-κB dimers composed of p65 and p50 subunits that translocate to the nucleus and bind to κB recognition sites in the promoter regions of inflammatory and immune genes, leading to their transcriptional activation. This response is primarily dependent on the catalytic subunit IKK-β (also known as IKK2), which carries out IκB phosphorylation. The non-canonical (alternative) pathway phosphorylates IKK-α homodimers, releasing RelB and lymphotoxin. It involves the upstream kinase NF-κB-inducing kinase (NIK), which processes p100 to p52 in response to certain members of the TNF family, such as TNF-β [Ibid., citing Sun, SC (2012) Immunol. Rev. 246:125-140]. This pathway can switch on a different set of genes and mediate immune functions that differ from the canonical pathway. Dominant-negative IKK-β inhibits most of the inflammatory functions of NF-κB, whereas inhibiting IKK-α only plays a role in response to limited stimuli and in certain cells, such as B lymphocytes. The non-canonical pathway is involved in immune system development and adaptive immune responses.The coactivator molecule CD40 expressed on antigen-presenting cells such as dendritic cells and macrophages activates the non-canonical pathway when it interacts with CD40L expressed on lymphocytes [Ibid., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol. 151:179-89].

[0162] As used herein, terms such as "occlusive", "occlusive", "occlusive" refer to a transdermal formulation that is applied to the skin using a support or otherwise related structure. For example, a topical formulation may be applied to the skin of a subject with the aid of a structure such as a backing member, bandage, or cover. A matrix patch is an example of an occlusive device. Conversely, the terms "non-occlusive" and "non-occlusive" may be used interchangeably, but refer to a transdermal formulation that is applied to the skin without the use of a support, backing, cover, or other related structure. For example, a transdermal formulation is applied to the skin in a free form, which is sufficient to achieve transdermal delivery of an active agent without the use of a structure such as a backing member. A gel formulation is an example of a non-occlusive composition, and other non-occlusive compositions include ointments, lotions, pastes, mousses, aerosols, and creams.

[0163] The term "penetration" and its various grammatical forms as used herein refer to the delivery of a substance through the skin.

[0164] As used herein, the term "penetration enhancer" refers to an agent known to accelerate the delivery of a substance through the skin.

[0165] The term "percutaneous absorption" refers to the absorption of a substance from the outside of the skin to a location below the skin, including into the bloodstream. The epidermis of human skin is highly relevant to the rate of absorption. Crossing the stratum corneum represents the rate-limiting step of percutaneous absorption. For example, the main steps involved in the percutaneous absorption of a drug include the establishment of a concentration gradient that provides the driving force for the movement of the drug across the skin, the release of the drug from the vehicle into the skin fraction, and the diffusion of the drug across the layer of the skin diffusion coefficient. The relationship of these factors to each other is summarized by the following equation: J=C veh ×K m .D / x [Formula VII] During the ceremony: J = Absorption rate C veh = drug concentration in vehicle K m = partition coefficient D = diffusion coefficient

[0166] There are many factors that affect the rate of percutaneous absorption of a substance. These are mainly: (i) concentration. The more concentrated the substance, the higher the absorption rate; (ii) the size of the skin surface area to which the drug is applied. The larger the contact area of ​​the skin to which the substance is applied, the higher the absorption rate; (iii) the anatomical application site. The thickness of the skin varies in various parts of the body. A thicker, more intact stratum corneum reduces the absorption rate of the substance. The stratum corneum in the facial area is much thinner than, for example, the skin of the palm of the hand. The structure of the facial skin and the thinness of the stratum corneum provide an area of ​​the body optimized for percutaneous absorption, allowing active agents to be delivered locally and systemically throughout the body; (iv) hydration. Hydration (meaning increasing the water content of the skin) swells the stratum corneum and increases permeability; (v) skin temperature. An increase in skin temperature increases permeability; (vi) composition. The composition of the compound and vehicle also determines the absorption rate of the substance. Most substances that are applied topically are incorporated into a base or vehicle. The vehicle selected for topical application can greatly affect absorption and can have beneficial effects on the skin itself. The factors that determine the choice of vehicle and the rate of transfer across the skin are the partition coefficient, molecular weight, and water solubility of the substance. The protein portion of the stratum corneum is most permeable to water-soluble substances, and the liquid portion of the stratum corneum is most permeable to lipid-soluble substances. Thus, substances that are both liquid-soluble and water-soluble can cross the stratum corneum more easily. See Dermal Exposure Assessment: Principles and Applications, EPA / 600 / 8-91 / 011b, January 1992, Interim Report-Exposure Assessment Group, Office of Health and Environmental Assessment, US Environmental Protection Agency, Washington, DC20460.

[0167] As used herein, the term "petechia" refers to tiny pinpoint to pinhead sized bleeding spots on the skin.

[0168] The term "pH adjusting agent" as used herein refers to a substance used to achieve the desired pH control in a formulation. Exemplary pH modifiers include acids (e.g., acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, lactic acid, phosphoric acid, sorbic acid, succinic acid, tartaric acid, basic pH modifiers (e.g., magnesium oxide, tribasic potassium phosphate, Eudragit® E), and pharma- ceutically acceptable salts thereof.

[0169] As used herein, the term "perianal" refers to being located in or affecting the area around the anal orifice.

[0170] As used herein, the term "plasminogen activator inhibitor-1 (PAI-1)" refers to a member of the superfamily of serine protease inhibitors (or serpins) and the primary inhibitor of both tissue-type and urinary-type plasminogen activators, two plasminogen activators capable of activating plasminogen. Plasminogen activators convert plasminogen to plasmin.

[0171] The term "polymer," as used herein, refers to any of a variety of compounds made from smaller identical molecules (called monomers) linked together. Polymers generally have a high molecular weight. The process by which molecules are linked together to form a polymer is called "polymerization."

[0172] As used herein, the term "preservative" refers to a substance added to a product to prevent degradation by microbial growth or undesirable chemical changes.

[0173] The term "promotion" as used herein in a medical context refers to processes that allow for increased control over the health of the vaginal, vulvar and / or perianal tissues and their determinants.

[0174] As used herein, the terms "psychoactive" and "psychotropic" refer to producing an effect on the mind or mental processes, such as a change in perception or behavior.

[0175] As used herein, the term "purification" and its various grammatical forms refer to the process of isolating or freeing from foreign, extraneous, or objectionable elements.

[0176] The term "pilin" as used herein refers to one of several protein interaction domains that are structurally related to, but distinct from, the CARD, TIR, DD, and DED domains.

[0177] The term "pyroptosis" as used herein refers to a form of programmed cell death associated with abundant proinflammatory cytokines, such as IL-1β and IL-18, produced by inflammasome activation.

[0178] The term "radiotherapy" as used herein refers to the use of high energy x-rays to kill cancer cells. Radiation may be used alone or with other treatments to effectively treat gynecological cancers. Radiation therapy can include both external beam radiation therapy using linear accelerators, and internal radiation therapy called brachytherapy. Internal radiation is given using a device called high dose rate (HDR) brachytherapy. Often referred to as an "implant," these HDR treatments involve placing a device inside the body very close to the cancer, loading the device with radiation, and delivering a high dose of radiation over a short period of time. Internal radiation is used when a dose of radiation needs to be delivered to a small area by getting the radiation as close as possible to the cancer cells. This allows a dose of radiation to be delivered to the cancer while normal nearby organs get a very low dose.

[0179] The term "reactive oxygen species" or "ROS", including free radicals and peroxides, refers to a class of molecules derived from the metabolism of oxygen and essentially present in all aerobic organisms. Most reactive oxygen species originate from endogenous sources as by-products of normal and essential metabolic reactions, such as energy production from mitochondria or detoxification reactions involving the cytochrome P-450 enzyme system in the liver.

[0180] The term "redox imbalance" as used herein refers to the overproduction of reactive oxygen or nitrogen species that overwhelms the cell's protective defense mechanisms. The consequences of this redox imbalance are lipid peroxidation, protein oxidation, DNA damage, and interference of reactive oxygen species with signaling pathways.

[0181] As used herein, the term "reduce" and its various grammatical forms refer to a decrease, reduction, attenuation, or diminution in degree, intensity, extent, size, amount, density, or number.

[0182] The term "rejuvenate" and its various grammatical forms as used herein refer to restoring a youthful state.

[0183] The term "repair" as used herein refers to restoring or restoring to a sound or good condition after damage or decay.

[0184] As used herein, the term "restore" and its various grammatical forms refer to returning to a previous or normal state, in order to recover or renew.

[0185] The term "skin" as used herein refers to the largest organ in the body, consisting of several layers. It plays an important role in biological homeostasis and is composed of the epidermis and dermis. The epidermis, consisting of several layers starting from the stratum corneum, is the outermost layer of the skin, and the innermost skin layer is the deep dermis. The skin has multiple functions, including thermoregulation, metabolic function (vitamin D metabolism), and immune function. Figure 1 shows the anatomical structure of the skin.

[0186] In humans, normal skin thickness is 1-2 mm, but there is considerable variation in various parts of the body. The relative proportions of epidermis and dermis also vary, with thicker skin being found in areas where one or both layers are thicker. For example, in the interscapular (between the shoulder blades) region of the back, where the dermis is particularly thick, the skin may exceed 5 mm, whereas on the eyelids it may be less than 0.5 mm. In general, the skin is thicker on the dorsal or extensor aspects of the body than on the ventral or flexor surfaces. However, this is not true of the hands and feet. The skin of the palms and plantars is thicker than any dorsal surface except the intrascapular region. The palms and soles have a characteristically thick epidermis in addition to a thick dermis.

[0187] The entire surface of the skin has many fine grooves, which run in a certain direction and cross each other to join small diamond-shaped or rectangular fields. These grooves correspond to similar grooves on the surface of the dermis, so that in cross section, the border between the epidermis and the dermis appears wavy. The thick skin of the palms and soles forms elongated ridges separated by parallel grooves, and on the fingertips they are arranged in complex loops, spirals (cyclops) and helices, giving each individual fingerprint its distinctive shape. These ridges are more pronounced in the areas where the epidermis is thickest.

[0188] Where there are lateral epidermal ridges, there are corresponding narrower processes on the skin surface called "interpapillary processes". The dermal papillae on either side of each interpapillary process project irregularly into the epidermis. On the palms and soles, and on other sensitive areas of the skin, the dermal papillae are numerous, tall, often branched, and of different heights (0.05 mm to 0.2 mm). Where mechanical demands are minimal and the epidermis is thin, such as on the abdomen and face, the papillae are low and few in number.

[0189] The epidermis provides the body's buffer zone against the environment. It provides protection from trauma, excludes toxins and microorganisms, provides a semipermeable membrane, and keeps vital fluids within a protective envelope. Traditionally, the epidermis is divided into several layers, two of which represent the most physiologically important. The basal cell layer, or stratum germinativum, is important because it is the main source of regenerative cells. In the process of wound healing, this is the area that most often undergoes mitosis. The epidermis, which includes the stratum granulosum and stratum granulosum, is the other area of ​​formation of normal epidermal barrier function.

[0190] The stratum corneum is an avascular multilayered structure that acts as a barrier to the environment and prevents transepidermal water loss. Recent studies have shown that enzymatic activity is involved in the formation of an acid mantle in the stratum corneum. Together, the acid mantle and stratum corneum reduce the permeability of the skin to water and other polar compounds, indirectly protecting the skin from microbial invasion. Normal superficial skin pH ranges from 4 to 6.5 in healthy individuals and varies with the area of ​​skin on the body. This low pH forms an acid mantle that enhances the skin barrier function.

[0191] Other layers of the epidermis below the stratum corneum include the stratum lucidum, stratum granulosum, stratum germinativum, and stratum basale. See Figure 2. Each contains living cells with specialized functions. For example, melanin produced by melanocytes in the epidermis is responsible for the color of the skin. Langerhans cells are involved in immune processing.

[0192] Skin appendages, including hair follicles, sebaceous and sweat glands, fingernails, and toenails, originate from the epidermis and project into the dermal hair follicles and sebaceous and sweat glands. They contribute epithelial cells for rapid re-epithelialization of wounds that do not penetrate the dermis (called partial wounds). Sebaceous glands are responsible for secretions that keep the skin lubricated, soft, and supple. They are most abundant on the face and sparse on the palms and soles. Sweat gland secretions control the pH of the skin and prevent skin infections. Sweat glands, cutaneous blood vessels, and small muscles in the skin (which cause goosebumps) control the temperature of the body's surface. Nerve endings in the skin include receptors for pain, touch, heat, and cold. Loss of these nerve endings increases the risk of skin breakdown by reducing the tissue's resistance to external forces.

[0193] The basement membrane separates and connects both the epidermis and the dermis. When epithelial cells within the basement membrane divide, one cell remains and the other migrates through the granular layer to the superficial stratum corneum. At the surface, cells die and form keratin. The dried keratin on the surface is called scale. Hyperkeratosis (thick layer of keratin) is often seen on the heels and indicates loss of sebaceous and sweat gland function if the patient is diabetic. The basement membrane atrophies with aging, and separation between the basement membrane and the dermis is one of the causes of skin fissures in the elderly.

[0194] The dermis, or dermis, is the vascular structure that supports and nourishes the epidermis. It also contains sensory nerve endings that transmit signals related to pain, pressure, heat, and cold. The dermis is divided into two layers: the superficial dermis and the deep dermis.

[0195] The superficial dermis is composed of extracellular matrix (collagen, elastin, and ground substance) and contains blood vessels, lymphatic vessels, epithelial cells, connective tissue, muscle, fat, and nerve tissue. The vascular supply of the dermis is responsible for nourishing the epidermis and regulating body temperature. Fibroblasts are responsible for producing the collagen and elastin components of the skin that give it fullness. Fibronectin and hyaluronic acid are secreted by fibroblasts. The structural integrity of the dermis plays a role in the normal function and youthful appearance of the skin.

[0196] The deep dermis lies above the subcutaneous fat. It contains a larger network of blood vessels and collagen fibers to provide tensile strength. It is also composed of fibroelastic connective tissue, which is yellow in color and mainly composed of collagen. Fibroblasts are also present in this tissue layer. The well vascularized dermis withstands pressure for a longer period of time than the subcutaneous tissue or muscle. Dermal collagen gives the skin its toughness. Dermal wounds, e.g., cracks or pustules, involve the epidermis, basement membrane, and dermis. Typically, skin injuries heal rapidly.

[0197] Substances are applied to the skin to induce one or more of four general effects: an effect on the skin surface, an effect within the stratum corneum, an effect requiring penetration into the epidermis and dermis, or a systemic effect that delivers a sufficient amount of a given substance through the epidermis and dermis into the vascular system to produce a therapeutic systemic concentration.

[0198] As used herein, the terms "soluble" and "solubility" refer to the property of being readily dissolved in a specified fluid (solvent). The term "insoluble" refers to the property of a material having minimal or limited solubility in a specified solvent. In a solution, the molecules of the solute (or dissolved substance) are evenly distributed among the molecules of the solvent.

[0199] According to the European Pharmacopoeia, the solubility of a compound in water at temperatures between 15° C. and 25° C. is defined as follows: [Table 2]

[0200] The term "solubilizer," as used herein, refers to a substance that allows a solute to dissolve.

[0201] A "solution" is generally considered to be a homogeneous mixture of two or more substances. It is often, but not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are evenly distributed among the molecules of the solvent.

[0202] The term "solvate," as used herein, refers to a complex formed by the binding of solvent molecules with molecules of solute.

[0203] As used herein, the term "solvent" refers to a substance that can dissolve another substance (called a "solute") to form a uniformly dispersed mixture (a solution).

[0204] The term "comfort" as used herein refers to the reduction of pain or discomfort.

[0205] As used herein, the term "stable" and its various grammatical forms refer to the ability of a particular formulation to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications.

[0206] The term "stabilizer," as used herein, refers to a chemical that tends to inhibit reactions between two or more other chemicals.

[0207] As used herein, the term "subject in need thereof" refers to a female subject susceptible to or experiencing a vaginal-vulvar or genitourinary symptom of trauma, injury or injury.

[0208] The term "suspension" as used herein refers to a dispersion (mixture) in which a finely divided species is combined with another species, the former being so finely divided and mixed that it does not settle rapidly. In everyday life, the most common suspensions are suspensions of solids in liquids.

[0209] The term "susceptible" as used herein refers to members of a population that are at risk. The term "susceptible population" as used herein refers to a subpopulation within the general population that is more likely to experience a disease.

[0210] The term "sustained release" (also referred to as "extended release") is used herein in its conventional sense to refer to a drug formulation that provides gradual release of drug over an extended period of time, preferably, although not necessarily, resulting in substantially constant blood levels of drug over the extended period of time.

[0211] As used herein, the term "synergy" refers to an interaction between two or more drugs that causes the total effect of the drugs to be greater than the sum of the individual effects of each drug.

[0212] As used herein, the term "symptom" refers to a sign or symptom of a disorder or disease, especially when experienced by an individual as a change from normal function, sensation, or appearance.

[0213] The term "therapeutic effect" as used herein refers to the outcome of treatment, which outcome is deemed desirable and beneficial. Therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of disease symptoms. Therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease symptoms.

[0214] As used herein, the terms "therapeutic amount," "beauty dose," or "effective amount" of one or more active agents refer to an amount sufficient to provide the intended benefit of treatment.

[0215] The term "tissue," as used herein, refers to a collection of cells that act together to perform a specific function. There are four basic tissues in the body: 1) epithelium, (2) connective tissue, including blood, bone, and cartilage, (3) muscle tissue, and 4) nervous tissue. In healthy tissue, cells remain in place and are attached to each other in structures that characterize the tissue and aid in its function.

[0216] Toll-like receptors Toll-like receptors (TLRs) are sensors of microorganisms present in the extracellular space. Some are cell surface receptors (e.g., TLR-1, TLR-2, TLR-5, TLR-6), while others (e.g., TLR3, TLR-7, TLR-8, TLR-9) are located within the membrane of endosomes and detect pathogens or their components that have been internalized into the cell by phagocytosis, receptor-mediated endocytosis or micropinocytosis. Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 88.

[0217] TLR-4, expressed by several types of immune system cells, including dendritic cells and macrophages, recognizes LPS from gram-negative bacteria by partially direct and partially indirect mechanisms. Systemic injection of LPS causes circulatory and respiratory collapse (shock) and systemic vascular permeability due to overwhelming secretion of cytokines, especially TNF-α. To recognize LPS, the ectodomain of TLR-4 first uses an accessory protein, MD-2, which binds to TLR-4 inside the cell and is necessary for both correct trafficking of TLR-4 to the cell surface and recognition of LPS. TLR-4 activation involves two other accessory proteins, LPS-binding protein, present in the extracellular fluid in blood and in tissues, and CD14, present on the surface of macrophages, neutrophils, and dendritic cells. CD14 can act as a phagocytic receptor by itself, but on macrophages and dendritic cells, it also acts as an accessory protein for TLR-4. Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 92.

[0218] Mammalian TLRs recognize molecules characteristic of bacteria, fungi, and viruses, including lipoteichoic acid from the cell wall of gram-positive bacteria and lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria. Although TLRs have limited specificity compared to the antigen receptors of the adaptive immune system, they can recognize elements of most pathogenic microorganisms and are expressed by many types of cells, including macrophages, dendritic cells, B cells, stromal cells, and certain epithelial cells. Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 88.

[0219] Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses by activating several different signaling pathways, each of which activates different transcription factors, which together lead to the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and the antiviral cytokines interferon alpha and beta. Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 92. The outcome of TLR activation can vary depending on the cell type in which it occurs. Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 95.

[0220] Signaling by mammalian TLRs is activated when binding of a ligand induces the formation of dimers or induces a conformational change in a preformed TLR dimer. All mammalian TLR proteins have a Toll-IL-1 receptor (TIR) ​​domain in their cytoplasmic tail that typically interacts with other T1R-type domains in other signaling molecules, and is also found in the cytoplasmic tail of the receptor for the cytokine interleukin-1-β. Ibid., 88. Dimerization brings the cytoplasmic T1R domains together and allows them to interact with the T1R domains of cytoplasmic adaptor molecules that initiate intracellular signaling. There are four adaptors used by mammalian TLRs: MyD88, MAL (also known as TIRAP), TRIF, and TRAM. The T1R domains of different TLRs interact with different combinations of these adaptors [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 92-93].

[0221] For example, TLR-3 interacts only with TRIF. Signaling by TLR-2 heterodimers (TLR-2 / 1 and TLR2 / 6) requires MyD88 / MAL. TLR-4 signaling uses both MyD8 / MAL and TRIF / TRAM, which are used during endosomal signaling by TLR-4. The choice of adaptor influences which of several downstream signals are activated by the TLR [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 94].

[0222] Signaling by most TLRs activates the transcription factor NFκB, several members of the interferon regulatory factor (IRF) transcription factor family via an alternative pathway, and members of the activator protein 1 (AP-1) family, such as c-Jun, through alternative signaling pathways involving mitogen-activated protein kinases (MAPKs). NFκB and AP-1 act primarily to induce the expression of proinflammatory cytokines and chemotactic factors [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 94].

[0223] Signaling pathways triggered by TLRs that use MyD88 TLR-7, TLR-8 and TLR-9 signal uniquely through MyD88. MyD88 has a T1R domain at its carboxy terminus that associates with the T1R domains of TLR cytoplasmic tails. At its amino terminus is a death domain that associates with similar death domains present in other intracellular signaling proteins. Both domains are necessary for signaling. The MyD88 death domain recruits and activates two serine-threonine protein kinases - IRAK4 (IL-1 receptor-associated kinase 4) and IRAK1 - through their death domains. The IRAK complex recruits enzymes that generate a signaling scaffold and uses this scaffold to recruit other molecules that are then phosphorylated by IRAK [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 94]. To form a signaling scaffold, the IRAK complex recruits the enzyme tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase that acts in concert with UBC13, an E2 ubiquitin ligase, and its cofactor Uve1A (together termed TRIKA1). The combined activity of TRAF-6 and UBC13 is to ligate one ubiquitin molecule to another protein, thereby generating a protein polymer. The polyubiquitin polymer can be extended with other proteins, including TRAF-6 itself, to generate polyubiquitin chains that function as scaffolds to bind other signaling molecules. The scaffold then recruits a signaling complex consisting of the polyubiquitin-binding adaptor proteins TAB1, TAB2, and the serine-threonine kinase TAK1. TAK1 is phosphorylated by the IRAK complex, and activated TAK1 propagates signaling by activating certain MAPKs, such as c-Jun terminal kinase (JNK) and mAPK14 (p38 MAPK), which then activate AP-1 family transcription factors that transcribe cytokine genes.

[0224] TAK1 also phosphorylates and activates the IκB kinase (IKK) complex, which consists of three proteins: IKKα, IKKβ, and IKKγ (also known as NEMO for NFκB essential modifier). NEMO binds polyubiquitin chains, bringing the IKK complex into close proximity with TAK1. TAK1 phosphorylates and activates IκB (inhibitor of κB), a cytoplasmic protein that constitutively binds to the transcription factor NFκB. NFκB contains two subunits, p50 and p65. Binding of IκB traps NFκB proteins in the cytoplasm. Phosphorylation by IKK induces degradation of IκB, which can release NFκB into the nucleus and drive transcription of genes for proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 94-95].

[0225] TLR-3, TLR-7, and the nucleic acid-sensing TLRs TLR-8 and TLR-9 activate members of the IRF family. IRF proteins reside in the cytoplasm and are inactive until phosphorylated on serine and threonine residues at their carboxy termini. They then translocate to the nucleus as active transcription factors. There are nine IRF family members, of which IRF3 and IRF7 are particularly important for TLR signaling and expression of antiviral type 1 interferons. In the case of TLR-3, expressed by macrophages and conventional dendritic cells, the cytoplasmic T1R domain interacts with the adaptor protein TRIF, which in turn interacts with the E3 ubiquitin ligase TRAF3, which generates a polyubiquitin scaffold similar to TRAF6. In TLR-3 signaling, this scaffold recruits a multiprotein complex containing the kinases IKKε and TBK1, which phosphorylate IRF3. TLR-4 also triggers this pathway by binding to TRIF, but the IRF3 response induced by TLR-4 is relatively weak compared to that induced by TLR-3.

[0226] For TLR-7 and TLR-9 signaling in plasmacytoid dendritic cells, the MyD88 T1R domain recruits the IRAK1 / IRAK4 complex, which can also physically associate with IRF7, which is highly expressed by plasmacytoid dendritic cells. This leads to IRF7 being phosphorylated by IRAK1, leading to the induction of type 1 interferon. Not all IRF factors regulate type 1 interferon genes. For example, IRF5 plays a role in the induction of proinflammatory cytokines.

[0227] NOD-like receptors (NLRs) Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are innate sensors that detect microbial products or cellular damage in the cytoplasm or activate signaling pathways and are expressed in cells that are routinely exposed to bacteria, such as epithelial cells, macrophages, and dendritic cells.

[0228] Some NLRs activate NFκB to initiate the same inflammatory responses as TLRs, whereas others trigger distinct pathways that induce cell death and production of inflammatory cytokines [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 96].

[0229] Subfamilies of NLRs can be distinguished based on the other protein domains they contain. For example, the NOD subfamily has an amino-terminal caspase recruitment domain (CARD) that is structurally related to the T1R death domain in MyD88 and can dimerize with CARD domains on other proteins to induce signaling. NOD proteins recognize fragments of bacterial cell wall peptidoglycan, but it is unclear whether they do so through direct binding or through accessory proteins [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 96]. NOD1 senses γ-glutamyldiaminopimelic acid (iE-DAP), a degradation product of peptidoglycan in Gram-negative and some Gram-positive bacteria, whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycan of most bacteria. Ibid. Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their functions are less well understood [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 96-98].

[0230] When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine-threonine kinase RIP2 (also known as RICK and RIPK2), which associates with the E3 ligases cIAP1, CIAP2, and XIAP, and its activity generates a polyubiquitin backbone that recruits TAK1 and IKK, leading to the activation of NFκB. NFκB then induces the expression of genes for enzymes involved in the production of inflammatory cytokines and NO [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 97].

[0231] Macrophages and dendritic cells express both TLF and NOD1 and NOD2 and are activated by both pathways. In epithelial cells, NOD1 may also function as a systemic activator of innate immunity. NOD2 is strongly expressed in intestinal Paneth cells, where it regulates the expression of potent antimicrobial peptides such as α- and β-defensins [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 97].

[0232] Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their functions are less well understood [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 96-98].

[0233] Another subfamily of NLR proteins, the NLRP family, has a pyrin domain instead of an amino-terminal CARD domain. Humans have 14 NLR proteins that contain a pyrin domain, of which NLRP3 (also known as NAPL3 or cryopyrin) is the best characterized. NLRP3 exists in an inactive form in the cytoplasm where its leucine-rich repeat (LRR) domain is thought to bind the head shock chaperone protein HSP90 and the co-chaperone SGT1. NRLP3 signaling is induced by reduced intracellular potassium, generation of reactive oxygen species, or disruption of lysosomes by particulate or crystalloid matter. For example, the death of nearby cells can activate the purinergic receptor P2X7, a potassium channel, releasing ATP into the extracellular space that would allow potassium ion efflux. A proposed model for ROS-induced NLRP3 activation involves the intermediate oxidation of sensor proteins collectively referred to as thioredoxins (TRXs). Typically, TRX proteins bind to thioredoxin-interacting proteins (TXNIPs). Oxidation of TRX by ROS causes dissociation of TXNIP from TRX. Free TXNIP can then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both cases, NLRP3 activation involves aggregation of multiple monomers via the leucine-rich repeat (LRR) and NOD domains, inducing signal transduction. Phagocytosis of particulate matter (e.g., the adjuvant alum) can result in lysosomal rupture and release of the active protease cathepsin B, which can activate NLRP3 [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 98-99].

[0234] NLR signaling, as exemplified by NLRP3, leads to the production of inflammatory cytokines and cell death through the formation of a multiprotein complex, the inflammasome. Activation of the inflammasome proceeds in several steps: (1) Aggregation of NLRP molecules triggers the autocleavage of procaspase I, which releases active caspase 1-the LRR domain of some NLRP3 molecules, or the aggregation of other NLRP molecules by specific triggering or recognition events, and induces the pyrin domain of NLRP3 to interact with an adaptor protein consisting of the pyrin domain, amino-terminal pyrin domain and carboxy-terminal CARD domain of ASC (also called PYCARD), with the pyrin domain at the center and the CARD domain facing outward to further drive the formation of polymeric ASC filaments. The CARD domain interacts with the CARD domain of the inactive protease procaspase 1, initiating its CARD-dependent polymerization into separate caspase 1 filaments. Active caspase-1 then carries out ATP-dependent proteolytic processing of inflammatory cytokines, particularly IL-1β and IL-18, to their active forms, inducing a form of cell death (pyroptosis) associated with inflammation due to the release of these inflammatory cytokines upon cell rupture [Janeway's Immunology, 9th Ed. 2017, Garland Science, New York, at 99-100].

[0235] The term "topical" as used herein refers to administration of the composition of the present invention at or just below the point of application. The terms "topical administration" and "topical application" as used herein are used interchangeably to refer to delivery of the composition to one or more surfaces, including epithelial surfaces. For example, the composition can be applied by pouring, dripping, or spraying in the case of a liquid, rubbing in the case of an ointment, lotion, cream, gel, etc., sprinkling in the case of a powder, spraying in the case of a liquid or aerosol composition, or any other suitable means. Topical administration generally produces a local effect rather than a systemic effect.

[0236] The term "treatment" or "treating" includes abolishing, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially ameliorating the clinical or aesthetic symptoms of a condition, substantially preventing the appearance of clinical or aesthetic symptoms of a disease, condition, or disorder, and protecting against harmful or bothersome symptoms. Treatment further refers to achieving one or more of: (a) reducing the severity of the disorder, (b) limiting the onset of symptoms characteristic of the disorder(s) being treated, (c) limiting the worsening of symptoms characteristic of the disorder(s) being treated, (d) limiting the recurrence of the disorder(s) in subjects who previously had the disorder(s), and (e) limiting the recurrence of symptoms in subjects who were previously asymptomatic for the disorder(s).

[0237] The term "trauma," as used herein, refers to an injury or wound to living tissue caused by an exogenous force.

[0238] The term "tumor necrosis factor alpha" or TNFα, as used herein, refers to a cytokine made by white blood cells in response to antigens or infection that induces necrosis (death) of tumor cells and has a wide range of proinflammatory effects. Tumor necrosis factor is also a multifunctional cytokine that affects lipid metabolism, coagulation, insulin resistance, and the function of endothelial cell-lining blood vessels.

[0239] As used herein, the term "vascular endothelial growth factor A" or "VEGFA" refers to a heparin-binding protein that exists as a disulfide-linked homodimer. This growth factor induces the proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis.

[0240] The term "viscosity" as used herein refers to the property of a fluid to resist forces tending to cause it to flow. The resistance is caused by intermolecular friction exerted when layers of fluid attempt to slide over one another. Viscosity can be of two types: dynamic (or absolute) viscosity and kinematic viscosity. Absolute viscosity or absolute viscosity coefficient is a measure of internal resistance. Dynamic (or absolute) viscosity is the tangential force per unit area required to move one horizontal surface relative to another at unit speed when maintained a unit distance by the fluid. Dynamic viscosity is usually expressed in poise (P) or centipoise (cP), with 1 poise = 1 g / cm 2 , and 1 cP = 0.01 P. Kinematic viscosity is the ratio of absolute or dynamic viscosity to density. Kinematic viscosity is usually expressed in Stokes (St) or centistokes (cSt), with 1 St = 10-4 m 2 / s, 1cSt=0.01St.

[0241] As used herein, the term "vagina" refers to the female genital canal that extends from the uterus to the vulva.

[0242] The term "Vaginal Health Index (VHI) score" refers to a tool that allows the determination of a final score that defines the degree of atrophy of the urogenital tract by assessing five parameters (vaginal elasticity, vaginal secretions, pH, epithelial mucosa, vaginal hydration) by assigning a single score to each parameter. The score can vary between 5 and 25, with a cutoff of <15 index for an atrophic vagina. [See Alvisi, S. et al. Medicina (2019) 55(10): 615].

[0243] The Vaginal Maturity Index is a score indicating the maturity of the tissue and measures the percentage of superficial, intermediate, and parabasal cells. [See Alvisi, S. et al. Medicina (2019) 55(10):615]. The Maturation Value (MV) is calculated as follows: MV = % superficial cells + (0.5 × % intermediate cells).

[0244] The term "vaginovulva" is meant to relate to the vagina and vulva.

[0245] As used herein, the term "vitality" refers to a characteristic that distinguishes living from non-living that is essential or necessary for the existence, continuance, or well-being of a full and healthy life.

[0246] The term "volume / volume percentage (v / v%)" refers to a measure of the concentration of a substance in a solution. It is expressed as the ratio of the volume of the solute to the total volume of the solution multiplied by 100.

[0247] As used herein, the term "vulva" refers to the female external genitalia consisting of the pubic bone, the labia majora and minora, the clitoris, the vagina and its glandular vestibule, and the urethral and vaginal openings.

[0248] The Vulvar Health Index (VHI) is a score that can be used to assess the vulva, including vulvar inflammation, muscle tissue contraction, pain on speculum insertion, and epithelial integrity. Scores can vary from 0 to 24, with a cutoff index of >8 for an atrophic vulva. [See Alvisi, S. et al. Medicina (2019) 55(10):615].

[0249] As used herein, the term "wound healing" or "wound repair" generally refers to the body's natural process of regenerating dermal or epidermal tissue. When an individual is injured, a complex series of biochemical events occurs to repair the damage, including hemostasis, inflammation, proliferation, and remodeling.

[0250] The term "wound healing agent" as used herein refers to an agent that promotes the complex process by which skin or other body tissues repair themselves after injury. In normal skin, the epidermis (top layer) and dermis (deep layer) form a protective barrier against the external environment. Thus, the term "wound healing agent" refers to any substance that facilitates the wound healing process.

[0251] As used herein, "wt %" or "weight percent" or "weight percent" or "wt / wt %" of a component, unless specified to the contrary, refers to the ratio of the weight of the component to the total weight of the composition in which it is included, expressed as a percentage.

[0252] Embodiment According to one aspect, the described invention provides a cosmetic composition formulated for topical application, comprising an aqueous gel, botanical ingredients, and a cosmetic composition stabilization system as described in U.S. Patent Application No. 16 / 867,370, which is incorporated herein by reference.

[0253] Water-based gel component background Hyaluronic acid (HA) is a member of a large family of glycosaminoglycans (GAGs) that are major components of the extracellular matrix. HA molecules consist of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-glycosidic bonds. [ka] This simple molecular unit has a molecular weight of 5 × 10 6 It forms long linear polymers up to 10 kDa. Long hyaluronan polymers have the ability to bind large amounts of water.

[0254] In its native form as a very long polymer, hyaluronic acid (also known as hyaluronan HA) is known as high molecular weight hyaluronic acid (HMWHA). Under certain conditions, it can be degraded into smaller fragments called low molecular weight HA ("LMWHA"). [Litwiniuk, M. et al. Wounds (2016) 28(3): 78-88, citing Aya, KL & Stern, R. Wound Repair Regen. (2014) 22(5): 579-93]. In somatic tissues, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2) are responsible for HA degradation. First, Hyal-2, a plasma membrane-like enzyme, degrades HA into fragments of molecules8 up to 20 kDa. These HA molecules are subsequently endocytosed and delivered to lysosomes, where further digestion is carried out by Hyal-1. [Ibid. citing Stern, R. & Jedrzejas, M. Chem. Rev. (2006) 106(30:818-39). In damaged tissue, free radicals can also break down HA polymers into smaller fragments. [Ibid. citing Longacre, MT et al. Ann. Surg. (1991) 213(4):292-96].

[0255] It is well documented that HMWHA exhibits anti-inflammatory and immunosuppressive properties, whereas LMWHA degradation products of HA can induce inflammation. [Ibid. citing Prevo, R. et al. J. Biol. Chem. (2001) 276(22):19420-30]. Small hyaluronan fragments have been shown to increase the expression and protein production of several cytokines, such as MMP-12, plasminogen activator inhibitor-1 (Id., Horton, MR et al. Am. J. Physiol. Lung Cell Mol. Physiol. (2000) 279(4):L707-15, citing Horton, MR, et al. J Immunol. (1999) 162(7):4171-76), macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant 1, keratinocyte chemoattractant, interleukin-8 (IL-8), and IL-12 by macrophages. [Ibid., citing Horton, MR, et al. J. Immunol. (1998) 160(6):3023-30; Hodge-DuFour, J. et al. J. Immunol. (1997) 159(5):2492-2500; McKee, CM et al. J. Clin. Invest. (1996) 98(10):1403-13]. CD44 is the major receptor for hyaluronan, but other receptors, such as TLRs, are also involved in HA signaling. [Ibid., citing Teder, P. et al. Science (2002) 296(5565):155-58]. LMWHA can bind to TLR receptors and initiate a signaling cascade that leads to the production of inflammatory cytokines and chemokines in various cell types in vitro. [Ibid. citing Litwiniuk, M. et al. Cent. Eur. J. Immunol. (2009) 34(4):247-51]. In immune cells from injured tissue, TLR2 and TLR4 activation by LMWHA has been shown to lead to the initiation of MyD88-dependent NFκB signaling cascade and pro-inflammatory cytokine gene expression.[Ibid., citing Jiang, D. et al. Nat. Med. (2005) 11(11): 1173-79, Suga, H. et al. J. Dermatol. Sci. (2014) 73(2): 117-24]. Induction of LMWHA by TLR-associated myeloid differentiation primary response gene 88 (MyD88) / NFκB signaling was also confirmed in breast tumor cells. Small HA fragments were shown to stimulate CD44 association with TLR2, TLR4 and MyD88, leading to NF-κB-specific transcriptional activation and expression of inflammatory cytokines Il-1β and IL-8 in human breast cell lines. Taken together, these reports suggest that LMWHA induces inflammation through activation of TLR receptors and initiation of MyD8 / NFκB signaling, which leads to the production of inflammatory cytokines and chemokines.

[0256] In physiological conditions, the activation of immune system cells is important for proper wound healing. In acute wounds, small hyaluronan fragments deposited at the site of injury activate the immune system to manage the breakdown of tissue integrity, whereas in chronic wounds, a constant and excessive inflammatory response actually impedes wound healing. [Ibid.]

[0257] LMWHA, which has strong antioxidant properties, exhibits protective effects against ROS, inhibits lipid peroxidation, and scavenges free radicals both in vitro and in vivo. [Ibid., citing Ke, C. et al. Food Chem. Toxicol. (2011) 49(10):2670-75].

[0258] The anti-inflammatory potential of HMWHA is well documented in osteoarthritis. HA is a fundamental component of normal synovial fluid, and because HA concentrations are reduced in osteoarthritis-affected joints, intra-articular injections of HMWHA have been used to treat osteoarthritis. For example, HMWHA was able to inhibit IL-1β expression in synovial cells in a rabbit model of osteoarthritis. [Ibid., citing Miki, Y. et al. Inflamm. Res. (2010) 59(6):471-77]. IL-1-dependent expression of MMP-1 and MMP-3 was reduced in human synovial cells by HMWHA treatment. A large study analyzed the effect of HMWHA on gene expression of various inflammatory cytokines by human fibroblast-like synoviocytes (FLS) in patients with early osteoarthritis. [Ibid. citing Wang, CT, et al. osteoarthritis Cartilage (2006) 14(12):1237-47] They reported downregulation of IL-8 and iNOS gene expression in unstimulated FLS and aggrecanase-2, as well as tumor necrosis factor alpha (TNFα) gene expression in IL-1 stimulated FLS. Blocking the CD44 receptor with anti-CD44 antibodies inhibited the downregulatory effect of HMWHA on gene expression. [Ibid. citing Bourguignon, LY et al. Cytoskeleton (Hoboken) (2011) 68(12):671-93].

[0259] The exact mechanism by which HMWHA interacts with TLR receptors, leading to inhibition of the inflammatory cascade, is unknown. HWHA could significantly reduce the expression of TLR4, TLR2, MyD88, and NF-κB in synovial cells in a mouse model of osteoarthritis. [Ibid., citing Campo, GM et al. Biochim. Biophys. Acta (2011) 1812(9):1170-81]. They also observed a decrease in the mRNA expression and protein production of TNFα, IL-1β, IL-17, MMP-13, and inducible nitrous oxide synthase genes in arthritic mice treated with HMWHA. [Ibid., citing Campo, GM et al. Biochim. Biophys. Acta (2011) 1812(9):1170-81], but only when HWHA was administered in the early inflammatory phase of osteoarthritis.

[0260] Examples of the antioxidant effects of HMWHA include the reduction of UVB-induced apoptosis and EDTA-induced oxidative damage to DNA [ibid., Bourguignon, LY et al. J. Biol Chem. (1997) 272(44): 27913-27918, citing Pauloin, T. et al., Mol. Vis. (2009) 15: 577-83], as well as the reduction of apoptosis and oxidative stress caused by benzalkonium chloride and sodium lauryl sulfate detergents, which are widely used in ophthalmic preparations [ibid., Pauloin, T. et al. Cornea (2009) 28(9): 1032-41, citing Pauloin, T. et al. Eur. J. Pharm. Sci. (20008) 34(4-5): 263-73]. The mechanism by which HMWHA reduces oxidative stress is not yet understood.

[0261] Many studies have shown that HA signaling plays a role in regulating angiogenesis, mainly by influencing endothelial cell behavior. Indeed, both HMWHA and LMWHA are potent regulators of angiogenesis. LMWHA stimulates vascular EC proliferation, migration, and tubule formation in vitro, as well as in various in vivo models of angiogenesis, while HMWHA exhibits antiangiogenic properties by inhibiting EC proliferation motility and sprouting formation. [Ibid., citing Toole, BP. Nat. Rev. Cancer (2004): 4(7): 528-39]. The exact molecular mechanisms that determine the pro- or antiangiogenic effects of different HA forms have not been fully elucidated. A context-dependent response to different forms of HA and a possible role of the microenvironment in this process have been suggested. In wound closure assays, the addition of CXCL12 to the culture medium significantly increased cell migration and induced faster wound closure. This effect was statistically enhanced when cells were preincubated with HMWHA. [Ibid. citing Fuchs, K. et al. Cell Death Dis. (2013) 4:e819]. In vitro studies showed that CXCR4 activation by CXCL12 was significantly increased in HUVECs pretreated with HMWHA, whereas preincubation with LMWHA blocked CXCL12 signaling in these cells.

[0262] In some embodiments, the sodium hyaluronate product (e.g., HMWHA, LMWHA, or both) is produced by biofermentation in a Steptococcus equi sub. Zooepidemicus strain. In some embodiments, the sodium hyaluronate product is obtained from a commercial source (e.g., Bloomage Biotechnology Corp., Ltd., Jinan, China). In some embodiments, the main components of the medium include wheat peptone, yeast extract powder, and glucose.

[0263] In some embodiments, the molecular weight of the HMWHA is in the range of 1,000 to 1,800 kDa (inclusive), i.e., at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa. In some embodiments, the molecular weight of the LMWHA is less than 10 kDa, i.e., at least 0.1 kDa to 10 kDa, at least 0.5 kDa to 10 kDa, at least 1 kDa to 10 kDa, at least 2 kDa to 10 kDa, at least 3 kDa to 10 kDa, at least 4 kDa to 10 kDa, at least 5 kDa to 10 kDa, at least 6 kDa to 10 kDa, at least 7 kDa to 10 kDa, at least 8 kDa to 10 kDa, or at least 9 kDa to 10 kDa.

[0264] In some embodiments, the finished product comprises about 0.10 to about 0.50 wt% (inclusive) LMWHA, i.e., 0.10 wt%, 0.15 wt%, 0.20 wt%, 0.25 wt%, 0.30 wt%, 0.35 wt%, 0.40 wt%, 0.45 wt%, or 0.50 wt% LMWHA, and about 0.50 to about 1.50 wt% (inclusive) HMHWA, i.e., 0.50 wt%, 0.60 wt%, 0.70 wt%, 0.80 wt%, 0.90 wt%, 1.0 wt%, 1.2 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, or 1.5 wt% HMHWA.

[0265] In some embodiments, the ratio of HMWHA to LMWHA ranges from 1:0.7 to 1:1 (inclusive), ie, 1:0.7, 0.8, 0.9, or 1.0.

[0266] plant raw material ingredients In some embodiments, the plant material component comprises a cannabinoid.

[0267] Cannabinoids are terpenophenolic secondary metabolites produced by cannabis. [Fischedick et al.,Phytochemistry 71:2058-73(2010)]. The cannabis strains used are dioecious (meaning they have male and female reproductive organs in separate individuals), and cannabinoids accumulate specifically in the unfertilized female inflorescence (meaning the complete flower head of the plant, including the stem, stalk, bracts, and flowers). [Ritchens et al.,PLoS ONE(2018)13:e0201119]. However, the synthesis and accumulation of cannabinoids occurs not only on the surface of the inflorescence, but also in the trichomes on the surface of the leaves. Happyana et al.,Phytochemistry 87:51-59(2013). Cannabinoids are also found in small amounts in the seeds, roots, and pollen of the plant. [Andre et al., Front.Plant Sci.7:19,oi:10.3389 / fpls.2016.00019(2016)]. Cannabinoids have also been found in plants of the genera Radula and Helichrysum. [Appendino et al., J.Nat.Prod.71:1427-30(2008)).

[0268] Over 140 cannabinoids have been reported, some of which are degradation products, and are generally classified into 11 subclasses [Berman et al., Sci. Rep. 8:14280(2018)]. The predominant compounds are Δ 9 -Tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), and cannabinodiolic acid (CBNDA). THCA is the primary cannabinoid in cannabis, while CBDA predominates in hemp.

[0269] The cannabis plant contains over 60 different active synthetic ligands for CB1 and CB2, of which Δ9-THC is the primary psychoactive molecule. [Kendall, DA, Yudowski, GA, Frontiers Cellular Neurosci. (2017) 10: 294] Exemplary cannabinoids include cannabichromene (e.g., cannabichromene (CBC), cannabichromenic acid (CBCA), cannabichromevarin (CBCV), cannabichromevarinic acid (CBCVA)), cannabicyclol (e.g., cannabicyclol (CBL), cannabicyclol acid (CBLA), cannabicyclovaline (CBLV)), cannabidiol (e.g., cannabidiol (CBD), cannabidiol monomethyl ether (CBDM), cannabidiolic acid (CBDA), cannabidiolcol (CBD-C1), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA)), cannabielsoin (e.g., cannabielsoinic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoinic acid A (CBEA-A)), cannabielsoinic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoinic acid A (CBEA-A)), cannabielsoinic acid B (CBEA-B), cannabielsoinic acid C (CBEA-C), cannabielsoinic acid C (CBEA-C), cannabielsoinic acid D (CBEA-D), cannabielsoinic acid E (CBEA-D), cannabielsoinic acid F (CBEA-F ... Bigerol (e.g., cannabigerol (CBG), cannabigerol monomethyl ether (CBGM), cannabigerolic acid (CBGA), cannabigerolic acid monomethyl ether (CBGAM), cannabigerovarin (CBGV), cannabigerovarinic acid (CBGVA)), cannabinol and cannabinodivalin (e.g., cannabinodivalin (CBND), cannabinodivalin (CBVD), cannabinol (CBN), cannabinol methyl ether (CBNM), cannabinol-C2 (CBN-C2), cannabinol-C4 (CBN-C4), cannabinolic acid (CBNA), cannabio-cool (CBN-C1), cannabivarin (CBV)), cannabidiol (e.g., 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxy-delta-6a-tetrahydrocannabinol, cannabidiol (CBT), cannabidiolvaline (CBTV)), Δ8-tetrahydrocannabinol (e.g., Δ8-tetrahydrocannabinol (Δ8-THC), Δ8-tetrahydrocannabinolic acid (Δ8-THCA)), Δ9-tetrahydrocannabinol (e.g., Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinol-C4 (THC-C4), Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinolic acid B (THCA-B), Δ9-tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9-tetrahydrocannabiolchol (THC-C1), Δ9-tetrahydrocannabiolcholic acid (THCA-C1), Cannabinoids include, but are not limited to, Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabivarinic acid (THCVA), and other cannabinoids (e.g., 10-oxo-Δ6a-tetrahydrocannabinol (OTHC), cannabichromanone (CBCF), cannabifuran (CBF), cannabiglendol, cannabilipsol (CBR), canbicitran (CBT), dehydrocannabifuran (DCBF), Δ9-cis-tetrahydrocannabinol (cis-THC), trihydroxy-Δ9-tetrahydrocannabinol (triOH-THC), 3,4,5,6-tetrahydro-7-hydroxy-α-α-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocin-5-methanol (OH-iso-HHCV)).

[0270] The cannabinoid profile of the cannabis plant, and the CBD / THC ratio of related plants, depends primarily on the genetic background of the plant, with each plant always belonging to its own unique chemical family throughout its life cycle. [Beutler et al., Bot. 32:387-94 (1978)]

[0271] In addition to cannabinoids, cannabis is also rich in bioactive terpenoids. Terpenoids or terpenes are aromatic compounds synthesized in trichomes. These compounds are typically present in several plant species, including Cannabis sativa, Mirabilis jalapa, Lithophragm glabrum, Cordia verbenacea, Eucalyptus globus, Syzygium aromaticum, Senna didymobotrya, Cymbopogon citratus, Pterodon emarginatus, Artemisia campestris, Lantana camara, Centella asiatica, Cyanthillium cinereum, Croton bonplandianus, and Citrus limon. [Goncalves et al.,Molecules 25:1567(2020)].

[0272] For example, B-caryophyllene and α-caryophyllene are major sesquiterpenes in cannabis. Booth et al., PLos ONE 12:e0173911(2017). Caryophyllene is a phytocannabinoid with strong affinity for CB2 but not CB1. Goncalves et al.(2020). Caryophyllene has been reported to be a repellent, antimicrobial, antibacterial, anticancer, antiproliferative, antifungal, AChE inhibitor, antioxidant, and anti-inflammatory agent. [Fidyt et al., Cancer Med.5:3007-17(2016), Sabulal et al., Phytochemistry 67:2469-73(2006), Su et al., Nat.Prod.Commun.11:845-48(2016), Sarvmeili et al. al.,Res.Pharm.Sci.11:476-83(2016), Memariani et al.,Oncol.Lett.11:1353-60(2016), Segat et al.,Neuropharmacology 125:207-19(2017), Bento et al. al., Am. J. Pathol. 178:1153-66 (2011), Gertsch et al., Proc. Natl. Acad. Sci. USA 105:9099-9104(2008), Alberti et al., J. Ethnopharmacol.155:485-94(2014)].

[0273] Limonene ((4R)-1-methyl-4-prop-1-en-2-ylcyclohexene) is the most common natural monoterpene found in nature and is found in hemp seeds, orange, lemon, and tangerine oils. Araujo-Filho et al., Neuroscience 358:158-69(2017). Limonene does not interact with either CB1 or CB2. Santiago et al., Cannabis Cannabinoid Res.4:165-76(2019). Limonene has been reported to be anti-inflammatory, gastroprotective, antinociceptive, antitumor, neuroprotective, antihyperalgesic, antidepressant, and anxiolytic. [Araujo-Filho et al. (2017), Al-Ghezi et al.,Front.Immunol.10:1921(2019), Sun, Altern.Med.Rev.J.Clin.Ther.12:259-64(2007), Shah et al.,Anim.Models Exp.Med.1:328-33(2018), d'Allessio et al.,Life Sci.92:1151-56(2013), de Almeida et al.,Inflammation 40:511-22(2017), do Amaral et al.,Biol.Pharm.Bull.30:1217-20(2007), Piccinelli et al. al., Nutr. Neurosci. 18:217-24 (2015)].

[0274] Linalool (3,7-dimethylocta-1,6-dien-3-ol) is a monoterpene compound present in several medicinal plants and fruits, including hemp, and is used in cosmetics and fragrance ingredients. Zhang et al., Enzym. Microb. Technol. 134:109462 (2020). Linalool has been reported to be anti-inflammatory, anti-cancer, anxiolytic, neuroprotective, UV-protective, and pain-reducing. Kim et al.,Int.Immunopharmacol.74:105706(2019), Sabogal-Guaqueta et al.,Biomed.Pharmacother.118:109295(2019),Harada et al.,Front.Behav.Neurosci.12:241(2018),Xu et al.,Life Sci.174:21-27(2017), Iwasaki et al.,World J.Gastroenterol.22:9765-74(2016), Gunsaeelan et al.,Photochem.Photobiol.Sci.Off.J.Eur.Photochem.Assoc.Eur.Soc.Photobiol.15:851-60(2016), Katsuyama et al. al., Biomed. Res. 33:175-81 (2012).

[0275] Terpineol (2-(4-methylcyclohex-3-en-1-yl)propan-2-ol) is a volatile monoterpene found in hemp oil, as well as cajuput oil, pine oil, and petitgrain oil. Goncalves et al. (2020). Terpineol has been reported to be antinociceptive, antifungal, anti-inflammatory, antidiarrheal, pain-reducing, memory-enhancing, algicidal, insect repellent, antiproliferative, and anticancer. de Oliveira et al.,Chem.Biol.Interact.254:54-62(2016), Chaudhari et al.,Food Chem.311:126010(2020), de Oliveira et al.,Basic Clin.Pharmacol.Toxicol.111:120-25(2012), dos Santos Negreiros et al. al.,Biomed.Pharmacother.110:631-40(2019), Gouveia et al.,Biomed.Pharmacother.105:652-61(2018), Parvardeh et al.,Iran.J.Basic Med.Sci.19:201-08(2016), Kim et al. al., Biosci.Biotechnol.Biochem.70:1821-26(2006), Nogueira et al. al.,Inflamm.Res.63:769-78(2014), Jing et al.,Bot.Stud.56:35(2015), Chen et al.,Ecotoxicol.Environ.Saf.167:435-40(2019),Wua et al. al., Nat.Prod.Commun.9:1515-18(2014), Villa-Ruano et al.,Chem.Biodivers.15:e1800354(2018), Hassan et al.,Anticancer Res.30:1911-19(2010).

[0276] γ-Terpinene (1-methyl-4-propan-2-ylcyclohexa-1,4-diene) is a monoterpene structurally similar to 1,8-cineole (eucalyptol) and is found in the essential oils of hemp and other plants including Eucalyptus (Myrtaceae), Cupressus cashmeriana, Lippia microphylla, Lavandula angustifolia, and Citrus myrtifolia. Goncalves et al. (2020). γ-Terpinene has been reported to be anti-inflammatory, antibacterial, analgesic, and anticancer. Djenane et al.,Food Sci.Technol.Int.17:505-15(2011), Ramalho et al.,Planta Med.81:1248-54(2015), da Silva Lima et al.,Eur.J.Pharmacol.699:112-17(2013),Guimaraes et al.,Phytother.Res.PTR 27:1-15(2013), Siveen et al., Can.J.Physiol.Pharmacol.89:691-703(2011), Ramalho et al., Axis.Planta Med.82:1341-45(2016), Baldissera et al. al., Exp. Parasitol. 162:43-48 (2016), Assmann et al. al., Biomed. Pharmacother. 103:1253-61 (2018).

[0277] Alpha-pinene is found in the essential oils of many aromatic plants, including hemp, Lavender angustifolia, Rosmarinus officinalis, and conifers. Begum et al., Acta Sci. Polonorum. Technol. Aliment. 12:61-73 (2013). Alpha-pinene has been reported to be an antioxidant, antibacterial, antitumor, hypnotic, anxiolytic, neuroprotective, cytoprotective, and antinociceptive agent. Zhao et al.,Chemotherapy 63:1-7(2018), Ibrahim et al.,Planta Med.85:431-38(2019), Nissen et al.,Fitoerapia 81:413-19(2010), Yang et al.,Mol.Pharmacol.90:530-39(2016), Satou et al. al.,Phytother.Res.PTR 28:1284-87(2014), Mercier et al.,Int.J.Occup.Med.Environ.Health 22:331-42(2009), Karthikeyan et al.,Life Sci.212:150-58(2018), Karthikeyan et al.,Life Sci.217:110-18(2019), Bouzenna et al., Biomed.Pharmacother.93:961-6(2017).

[0278] β-Pinene is found in many plant essential oils and can be obtained commercially by distillation or conversion to α-pinene. Iseppi et al., Molecules 24:2302(2019). β-Pinene has been reported to be antibacterial, antioxidant, anti-immobilizing, and anti-adhesive. Mahajan et al.,Environ.Sci.Pollut.Res.Int.26:456-63(2019), Guzman-Gutierrez et al.,Life Sci.128:24-29(2015), Astani et al.,Iran.J.Microbiol.6:149-55(2014), de Macedo Andrade et al. al.,Curr.Top.Med.Chem.18:2481-90(2018), Jia et al.,Antimicrob.Agents Chemother.46:947-57(2002), da Silva et al.,Molecules 17:6035-16(2012).

[0279] β-elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane) is a derivative, which may arise due to oxidation or thermal or UV-induced rearrangements during processing or storage. Booth et al. (2017). β-elemene has been reported to be an anticancer agent. Deng et al.,Phytother.Res.PTR 33:2248-56(2019), Wu et al.,Exp.Ther.Med.13:3153-57(2017), Fang et al.,Oncol.Lett.15:3957-64(2018), Cai et al. al.,Oncol.Lett.16:6019-25(2018), Liu et al.,Oncol.Rep.32:2635-47(2014), Li et al.,Anticancer Res.33:65-76(2013), Wei et al.,Oncol.Rep.37:3159-66(2017), Yoshida et al. al.,Lab.Investig.J.Tech.Methods Pathol.93:1184-93(2013), Liu et al.,Biomed.Pharmacother.95:1789-98(2017), Zhang et al.,Int.Immunopharmacol.10:738-43(2010).

[0280] β-Ocimene (3,7-dimethyl-1,3,6-octatriene) is an acyclic monoterpene that functions as a chemical cue to attract natural enemies of phytophagous insects in some plant species. Booth et al. (2017). β-Ocimene has been reported to be an antitumor, antifungal, and anticonvulsant. Bomfim et al., Basic Clin. Pharmacol. Toxicol. 118:208-13 (2016), Sayyah et al., J. Enthnopharmacol. 94:283-87 (2004).

[0281] Camphene (2,2-dimethyl-3-methylidenebicyclo(2.2.1)heptane) is a cyclic monoterpene present in cannabis inflorescences at low potency but abundant in Thymus vulgaris oil (Goncalves et al. (2020)). Camphene has been reported to be expectorant, antispasmodic, and antibacterial. Baser et al., Handbook of Essential Oils: Science, Technology, and Applications; CRC Press / Taylor & Francis: Boca Raton, FL (2010), Feng et al., Environ.Sci.Pollut.Res.Int.26:16157-65 (2019), Benelli et al. al., Ecotoxicol.Environ.Saf.148:781-86(2018), Benelli et al.,Environ.Sci.Pollut.Res.Int.25:10383-91(2018).

[0282] Nerolidol ((6E)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol; perviol) is an acyclic sesquiterpene alkene alcohol common to citrus peel, Piper claussenianum, Baccharis dracunculifolia, and cannabis. Baldissera et al., Naunyn-Schmiedeberg's Arch. Pharmacol. 391:753-59(2018). Nerolidol has been reported to be antibacterial and anti-inflammatory. Alonso et al.,Biochim.Biophys.Acta Biomembr.1861:1049-56(2019), Zhang et al.,Phytother.Res.PTR 31:459-65(2017), Iqubal et al.,236:116867(2019), Iqubal et al. al.,Eur.J.Pharmacol.863:172666(2019).

[0283] Euphorbia niger, a tetracyclic triterpene and minor cannabis component, is usually extracted in an alcohol preparation. Pellati et al., Biomed. Res. Int. 2018:1691428 (2018). Euphorbia niger has been reported to be anti-cancer and anti-inflammatory. Betancur-Galvis et al.,Mem.Inst.Oswaldo Cruz 97:541-46(2002), Prinsloo et al.,J.Ethnopharmacol.210:133-55(2018),Mazior et al.,Fur Naturforschung.Cjournal Biosci.66:360-66(2011),Silva et al. al.,Exp.Ther.Med.16:557-66(2018), Cruz et al.,Phytomedicine:Int.J.Phytother.Phytopharm.47:105-112(2018),Wang et al.,Mol.Med.Rep.8:1279-85(2013),Silva et al.,Investig.New Drugs 37:223-37(2019).

[0284] In plants, terpenoid compounds synthesized alongside phytocannabinoids are important volatile components responsible for the characteristic odor of plants and also serve various organic functions, such as insect repellent, repellent against herbivore attack, and attractive to pollinators. The unique chemical abundance of specific terpenoids is predicted to be related to chemotype and species-level taxa, so hemp terpenoid composition provides information about the origin of the plant. Fischedick et al. (2010). An analysis of 72 hemp strains showed that the total terpenoid content ranged between 0.6 and 3.3%, while the total cannabinoid content ranged between 12.6 ± 31.5%. [Ritchens et al. (2018) PLoS ONE (2018) 13: e0201119].

[0285] Many methods are known for extracting cannabinoids and terpenoids from hemp. Sonication is the most common and is the method recommended by both the United Nations Office on Drugs and Crime (UNDOC) and the American Herbal Pharmacopoeia (AHP). Giese et al., J.AOAC Int. 98:1503-22 (2015). The AHP monograph method recommends first drying and powdering the sample, and requires a separate moisture measurement to accurately assess the content in the initial inflorescence. It should also be noted that such drying and grinding alters the terpenoid content. Giese et al. (2015), Swift et al., PLoS ONE 8:e70052 (2013). Sonication has three notable limitations. Injection of hemp extract for analysis by gas chromatography usually results in decarboxylation at the injection port, and therefore only decarboxylated phytocannabinoids are directly measured by these techniques. Derivatization of metabolite extracts by silylation allows for the measurement of both acidic and neutral phytocannabinoids, but complete derivatization yields are difficult to obtain and quantification is unreliable. It has been suggested that phytocannabinoids may undergo thermal degradation (oxidation, isomerization) in the injection port and on the column. Berman et al. (2018).

[0286] Most cellular cannabinoid effects are mediated by two G protein-coupled receptors (GPCRs), CB1 and CB2. CB1 receptors are present at very high levels in the brain and in lower amounts more widely, and mediate most of the psychoactive effects of cannabinoids. CB2 receptors are more restricted in distribution, being found on certain immune cells and neurons. Mackie, J. Neuroendocrinol. 20(Supp.1):10-14(2008). Cannabinoids mediate both inhibitory and stimulatory effects on the immune system by regulating cytokine expression. [Raduner, S. et al., J. Biol. Chem. (2006) 281 (20): 14192-14206, Klein, T., et al. J. Leukocyte Biol. (2003) 74: 486-96, Croxford, JLand Yamamura, t J Neuroimmunol (2005) 166: 3-18]. Other GPCRs, ion channels, and nuclear receptors also interact with cannabinoids. [Zou et al., Int. J. Mol. Sci. 19: 833 (2018)]. N-arachidonoyl-ethanolamine and 2-arachidonoylglycerol are endogenous agonists of cannabinoid receptors. Zou et al. (2018). A third putative CB receptor, GPR55, shares only 13.5% sequence identity with CB1 and 14.4% with CB2. [Lauckner et al., Proc. Natl. Acad. Sci. USA 105:2699-2704(2008)]. GPR55 shares some ligands with CB1 and CB2 and has additional agonist ligands with novel chemotypes. [Kotsikorou et al., Biochemistry 52:9456-69(2013)].

[0287] In addition to cannabinoids, endogenous agonists of CB1 and CB2 (and possibly GPR55) include arachidonoylethanolamide (anandamide), 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether (norazine ether). [Pertwee et al., Prostaglandins Leukot. Essent. Fatty Acids 66:101-21(2002)]. There are also three classes of synthetic agonists: classical, bicyclic, and aminoalkylindole cannabinoids similar to THC. [Hourani et al., Brain Neurosci. Adv. 2:1-8(2018)]. Many of these synthetic cannabinoids are much more potent than THC, and they too have demonstrated greater efficacy. Selective synthetic agonists for CB1 and CB2 include SR141716A, LY320135, SR144528, 6-iodopravadrine (AM630), nabilone, CP55940, and R-(+)-WIN55212-2. Pertwee et al. (2002), Hourani et al. (2018). SR141716A and LY320135 are highly selective for CB1, and SR144528 and AM630 are highly selective for CB2. Pertwee et al. (2002).

[0288] Both phytocannabinoids and synthetic cannabinoids can directly affect the endocannabinoid system through a variety of pharmacological mechanisms, including agonism, antagonism, and allosteric modulation [see Bonn-Miller et al., Int. Rev. Psychiatry (2018) 30:277-84].

[0289] Oral formulations of synthetic cannabinoids are also available commercially. For example, Nabilone is a synthetic cannabinoid sold as CESAMET® in Canada, the United States, the United Kingdom, and Mexico. Nabilone is formulated as a capsule suitable for oral administration. NAMISOL® is approved for use as an antiemetic and analgesic for neuropathic pain. SAVITEX® is a mouth spray containing THC and CBD. US8808734. It is approved for the treatment of spasticity due to multiple sclerosis and as an adjunct analgesic in patients with advanced cancer. [Paudel et al., Drug Dev. Indus. Pharm. 36:1088-97 (2010). Administration of synthetic cannabinoid formulations has fewer undesirable side effects than THC. See US8808734.

[0290] The bioavailability of orally ingested pharmaceutical substances depends on the extent to which the pharmacologic active substance is absorbed from the intestinal environment across the intestinal mucosa. Lipophilic pharmaceutical substances are generally poorly absorbed from the intestinal environment, especially due to their poor solubility and / or dispersibility in water. The bioavailability of orally ingested pharmaceutical substances further depends on the substance's susceptibility to the so-called first-pass effect. Substances absorbed from the intestine must first pass through the liver before being distributed throughout the body, where they can be quickly metabolized. CBD is generally assumed to be fairly susceptible to first-pass hepatic metabolism. The oral bioavailability of CBD is low and unpredictable, and CBD is unstable. [Zhornitsky et al., Pharmaceuticals (2012) 5: 529-52, Poortman et al., Forensic Sci. Int. (1999) 101: 1-8].

[0291] Cannabinoids are lipophilic substances known to be poorly soluble in water (<1 μg / mL). As an example, CBD is soluble in ethanol (36 mg / mL) and dimethyl sulfoxide (DMSO) (60 mg / mL). CBD is highly lipophilic and undergoes first-pass metabolism after oral administration. [Nichols, JM, Kaplan, BLF. Cannabis and Cannabinoid Res. (2018) doi:10.1.089 / can.2018.0073, citing Samara, E. et al. Drug Metab. Dispos. (1988) 16:469-72]. Data to date overwhelmingly indicate that CBD is immunosuppressive and anti-inflammatory. [Ibid.] The identity of the receptors by which CBD acts in the immune system and the cell types expressing the receptors that mediate CBD effects are unknown. [Ibid.] The effects of CBD are known to be mediated by activation of CB1, CB2, the transient receptor potential V1 (TRPV1) also known as the vanilloid receptor, the adenosine A2A receptor, and the PPAR-γ receptor, blockade of the GPR55 receptor, and inhibition of fatty acid amide hydrolase (FAAH). [Ibid.] The effects of CBD on the immune response may involve innate or adaptive responses. [Ibid.] Targets of inhibition include cytokines such as TNF-α, IFN-γ, IL-6, IL-1β, IL-2, IL-17A, and chemokines such as CCL-2. [Ibid.] The overall mechanism of CBD involves direct inhibition of target cells such as effector T cells, innate, and microglial cells through the inhibition of kinase cascades and various transcription factors.

[0292] However, data examining the effects of CBD on various T cell subsets are limited. Results suggest that B cells may be targets for suppression by CBD, but only a few studies identify B cells as targets of CBD. Direct suppression of target cells also includes induction of IκB, which may contribute to reduced NF-κB activity. [Ibid.] Involvement of regulatory cell induction by CBD is also a major part of the mechanism by which CBD controls immune responses, and CBD has been shown to induce regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), the latter being innate myeloid cells with the ability to suppress T cell responses [Ibid., see Gabrilovich, D., Nagaraj, S. Nat. Rev. Immunol. (2009) 9(3):162-74]. CBD-induced apoptosis is likely a mechanism in many target cells. Nichols, JM, Kaplan, BLF. Cannabis and Cannabinoid Res. (2018)doi:10.1.089 / can.2018.0073]

[0293] EPIDIOLEX® is the first FDA-approved CBD medication (Greenwich Biosciences Inc., Carlsbad, CA) approved for use in patients aged 2 years and older with Dravet syndrome or Lennox-Gastaut syndrome. Four randomized, double-blind, multicenter clinical trials evaluated the use of CBD in patients with Dravet syndrome or Lennox-Gastaut syndrome for efficacy and safety in convulsive and hypotensive seizures, respectively. All studies showed a significant absolute reduction in seizure frequency. Devinsky et al., N. Engl. J. Med. (2017) 376: 2011-20, Devinsky et al., N. Eng. J. Med. (2018) 378: 1888-97, Thiele et al., Lancet (2018) 1085-96).

[0294] In some embodiments, the cannabinoid is derived from hemp. In some such embodiments, the cannabinoid is non-psychoactive. In some embodiments, the cannabinoid is CBD. In some embodiments, the cannabinoid is not full-spectrum CBD, i.e., does not contain all the cannabinoids found in the natural cannabis plant. In some embodiments, the cannabinoid is decarboxylated CBD (see formula IX below). [ka]

[0295] In some embodiments, the isolate comprises about 20% decarboxylated CBD. In some embodiments, the isolate comprising about 20% decarboxylated CBD is in the form of a THC-free nano-infused water-soluble powder. In some embodiments, the isolate comprising about 20% decarboxylated CBD is obtained from a commercial source (e.g., Global Cannabinoids, Las Vegas, NV). In some embodiments, the isolate comprising about 20% CBD comprises less than 1% cannabidivarin (CBDV), which has a molecular structure similar to CBD, but instead of having a pentyl chain, it has a propyl chain. In some embodiments, the decarboxylation is performed by heating to no more than 300°F, for example, 200°F (93°C) for 75 minutes, 225°F (107°C) for 50 minutes, or 250°F (121°C) for 30 minutes.

[0296] An exemplary analysis of decarboxylated CBD in the form of a THC-free nano-infused water soluble powder is shown below (LOQ=limit of quantification). [Table 3]

[0297] Results reported are based on sample weight at the appropriate moisture content for that sample.Unless otherwise stated, all quality control samples perform within the specifications established by the laboratory.

[0298] In some embodiments, total CBD can be represented by the formula CBDa*0.877+CBD and total THC can be represented by the formula THCa*0.877+Δ9-THC+Δ8THC. In some embodiments, the powder contains one or more inactive ingredients, such as tapioca maltodextrin.

[0299] In some embodiments, the finished product comprises between 1.0% and about 5.0% by weight (inclusive), i.e., at least 1.0%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2.0%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3.0%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4.0%, at least 4.25%, at least 4.5%, at least 4.75%, or at least 5% by weight of the 20% CBD-THC free nano-infused water soluble powder.

[0300] Cosmetic composition stabilization system In some embodiments, the cosmetic composition stabilizing system comprises an effective amount of an arginine component. In some embodiments, the arginine component comprises i) arginine, or a conjugate, or an analog thereof, ii) an organic acid, a conjugate, or an analog thereof, and iii) a solvent. As used herein, the phrase "arginine, a conjugate, or an analog thereof" is referred to as an "arginine compound." As used herein, the phrase "organic acid, a conjugate, or a derivative thereof" is referred to as an "organic acid compound." In some embodiments, the arginine component comprises i) an arginine compound, ii) an organic acid compound, and iii) a solvent.

[0301] In some embodiments, the cosmetic composition stabilizing system has a preservative effect, a solubilizing effect, a stabilizing effect, a neutralizing or antimicrobial effect, a moisturizing and / or healing effect on the composition, or any combination thereof. For example, in some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition and / or a solubilizing effect on the active agent. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing and / or neutralizing effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, stabilizing, and / or a neutralizing, and / or antibacterial effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing, stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing, exfoliating, and / or healing cosmetic / therapeutic effect.

[0302] In some embodiments, the arginine compound is arginine, or its salts, conjugates, or analogs.For example, arginine can be D, L-arginine, D-arginine, L-arginine, alkyl (ethyl, methyl, propyl, isopropyl, butyl, isobutyl, t-butyl) esters of arginine, and conjugates and analogs thereof.

[0303] In some embodiments, the arginine conjugate may be a conjugate acid or a conjugate base, for example, the arginine conjugate may be arginine, or an arginate.

[0304] In some embodiments, the arginine compound may be used in any suitable amount. For example, the arginine compound may be present in the arginine component at least about 1.0% by weight, at least about 5.0% by weight, at least 10.0% by weight, at least about 15.0% by weight, at least 20.0% by weight, at least 25.0% by weight, at least 30.0% by weight, at least 35.0% by weight, at least 40.0% by weight, at least 45.0% by weight, at least 50.0% by weight, at least 55.0% by weight, at least 60.0% by weight, at least 65.0% by weight, at least 70.0% by weight, at least 75.0% by weight, at least 80.0% by weight, at least 85.0% by weight, at least 90.0% by weight, and about 95.0% by weight of the total weight of the arginine component. Exemplary amounts include 10.0% to about 70.0% by weight (inclusive) based on the total weight of the arginine component, about 20% to about 60% by weight (inclusive) based on the total weight of the arginine component, and about 30% to about 50% by weight (inclusive) of the total weight of the arginine component.

[0305] In some embodiments, the organic acid compound can be an organic acid, its conjugate, or its analog. In some embodiments, the organic acid can be any suitable organic acid. For example, the organic acid can be substituted and unsubstituted aliphatic (saturated and unsaturated) and aromatic acids. The organic acid can have one or more functional groups as a substituent, such as alkyl, alkenyl, alkynyl, halogen, hydroxy, carbonyl, carboxylic acid, aldehyde, ester, amide, carbonate, carbamate, ether, amino, cyano, isocyano, oxy, oxo, thia, aza, azide, imine, nitro, nitrate, nitroso, nitrosoxy, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfhydryl, sulfonyl, phosphino, where each of the alkyl, alkenyl, alkynyl, and amino groups can themselves be optionally substituted with one or more of the aforementioned functional groups. In some embodiments, some functional groups, such as hydroxy, impart or enhance properties to the acid that are suitable for the present compositions, such as hygroscopic properties, hi some embodiments, the organic acid comprises multiple carboxylic acid groups.

[0306] In some embodiments, the organic acid is a hydroxy acid, in some embodiments, the hydroxy acid is an alpha-hydroxy acid (AHA), beta-hydroxy acid (BHA), gamma-hydroxy acid (GBA), omega-hydroxy acid, monohydroxy acid (MHA), polyhydroxy acid (PHA) / polycarboxyhydroxy acid (PCHA), aliphatic hydroxy acid (AlHA), aromatic hydroxy acid (ArHA), aliphatic hydroxy acid (AAHA), hydroxy fatty acid, etc.

[0307] In some embodiments, the hydroxy acid is an alpha-hydroxy acid (AHA). The alpha (α) carbon in an organic molecule refers to the first carbon atom that is bonded to a functional group such as a carbonyl. In some embodiments, the AHA is an alkyl AHA, an aryl alkyl AHA, or a polycarboxyl AHA.

[0308] In some embodiments, the organic acid component comprises anisic acid, also known as 2-methoxybenzoic acid, o-anisic acid, 579-75-9, o-methoxybenzoic acid, 2-anisic acid, O-methylsalicylic acid, benzoic acid, 2-methoxy-, salicylic acid methyl ether, 2-methoxybenzoic acid, 529-75-9, molecular formula CHO.

[0309] In some embodiments, the organic acid component comprises levulinic acid, also known as 4-oxopentanoic acid; 123-76-2; laevulinic acid; pentanoic acid, 4-oxo-4-oxovaleric acid; levulinic acid; 3-acetylpropionic acid; 4-ketovaleric acid; LEVA; molecular formula C5H8O3.

[0310] In some embodiments, the organic acid component comprises mandelic acid, also known as 17199-29-0, (S)-(+)-mandelic acid, (S)-mandelic acid, (S)-2-hydroxy-2-phenylacetic acid, L-mandelic acid, L-(+)-mandelic acid, S-(+)-mandelic acid, (2S)-2-hydroxy-2-phenylacetic acid, UNII-L0UMW58G3T, L(+)-mandelic acid, molecular formula CHO.

[0311] In some embodiments, the organic acid component comprises salicylic acid, also known as 2-hydroxybenzoic acid, 69-72-7, o-hydroxybenzoic acid, 2-carboxyphenol, o-carboxyphenol, Rutranex, Salonil, Retarder W, Keralyt, with the molecular formula C7H6O3 or HOC6H4COOH.

[0312] In some embodiments, the organic acid component comprises sorbic acid, also known as 110-44-1, 2,4-hexadienoic acid, (2E,4E)-hexa-2,4-dienoic acid, 2E,4E-hexadienoic acid, Panosorb, Sorbistat, 2-propenylacrylic acid, trans,trans-sorbic acid, hexadienoic acid, molecular formula CHO or CHCH=CHCH=CHCOOH.

[0313] In some embodiments, the organic acid component comprises benzoic acid, also known as 65-85-0, dolacyl acid, benzenecarboxylic acid, carboxybenzene, benzeneformic acid, phenylformic acid, benzenemethanoic acid, phenylcarboxylic acid, Retardex, with the molecular formula C7H6O2 or C6H5COOH.

[0314] In some embodiments, the organic acid component includes ferulic acid, also known as trans-4-hydroxy-3-methoxycinnamic acid, and trans-ferulic acid, with the molecular formula HOC6H3(OCH3)CH=CHCO2H.

[0315] In some embodiments, the organic acid component includes 3,5-dimethoxy-4-hydroxybenzoic acid, 4-hydroxy-3,5-dimethoxy-benzoic acid, and syringic acid, also known as gallic acid 3,5-dimethyl ether, molecular formula HOC6H2(OCH3)2CO2H.

[0316] In some embodiments, the organic acid component is anisic acid, levulinic acid, mandelic acid, salicylic acid, sorbic acid, benzoic acid, ferulic acid, and syringic acid, e.g., anisic acid and levulinic acid, anisic acid and mandelic acid, anisic acid and salicylic acid, anisic acid and sorbic acid, anisic acid and benzoic acid, anisic acid and ferulic acid, anisic acid and syringic acid, levulinic acid and mandelic acid, levulinic acid and salicylic acid, levulinic acid and sorbic acid, levulinic acid and benzoic acid, levulinic acid and ferulic acid, levulinic acid and syringic acid, levulinic acid and mandelic acid, levulinic acid and salicylic acid, levulinic acid and sorbic acid, levulinic acid and benzoic acid, levulinic acid and ferulic acid, levulinic acid and syringic acid, levulinic acid and mandelic acid, levulinic acid and salicylic acid, levulinic acid and sorbic acid, levulinic acid and ferulic acid, levulinic acid and syringic acid, levulinic acid and ferul ... The composition comprises two organic acids selected from the group consisting of phosphoric acid and syringeic acid, mandelic acid and salicylic acid, mandelic acid and sorbic acid, mandelic acid and benzoic acid, mandelic acid and ferulic acid, mandelic acid and syringeic acid, salicylic acid and sorbic acid, salicylic acid and benzoic acid, salicylic acid and ferulic acid, salicylic acid and syringeic acid, sorbic acid and benzoic acid, sorbic acid and ferulic acid, sorbic acid and syringeic acid, benzoic acid and ferulic acid, benzoic acid and syringeic acid, ferulic acid and syringeic acid.

[0317] In some embodiments, organic acids, such as a combination of the two exemplified above (e.g., levulinic acid and anisic acid), when used with glyceryl caprylate / caprate, form a broad-spectrum preservative that is effective against microbial contaminants, such as bacteria, yeasts, and molds. Without being limited to any particular theory, a stabilization system that includes glyceryl caprylate / caprate allows greater penetration of both acids into the cell walls of the target organism, which means that the overall concentration of acid in the formulation is lower, and the concentration of free acid required for full-spectrum preservation of the product is reduced. This, in turn, increases the effectiveness in finished formulations that are difficult at physiological pH, and reduces the need for additional ingredients in the finished formulation without drastic adjustment of the final pH of the finished formulation.

[0318] In some embodiments, organic acid compounds include any derivatives of the organic acid, for example derivatives of organic acids that revert to their acid form when contacted with water, including, but not limited to, readily hydrolyzable anhydride, mixed anhydride, and ester derivatives of organic acids.

[0319] In some embodiments, the organic acid compound may be used in any suitable amount. For example, the organic acid compound may be present in the arginine component as about 1.0 wt%, 5.0 wt%, 10.0 wt%, 15.0 wt%, 20.0 wt%, 25.0 wt%, 30.0 wt%, 35.0 wt%, 40.0 wt%, 45.0 wt%, 50.0 wt%, 55.0 wt%, 60.0 wt%, 65.0 wt%, 70.0 wt%, 75.0 wt%, 80.0 wt%, 85.0 wt%, 90.0 wt%, and about 95.0 wt% of the total weight of the arginine component. Exemplary amounts include about 10.0 wt% to about 70.0 wt% based on the total weight of the arginine component, about 20 wt% to about 60 wt% based on the total weight of the arginine component, and about 30 wt% to about 50 wt% of the total weight of the arginine component.

[0320] In some such embodiments, the pH is high enough to keep the acid in solution and low enough to keep glyceryl caprylate / caprate from hydrolysis. For example, the pH of the raw cosmetic composition stabilizing system in water containing glyceryl caprylate / caprate can range from pH 4.1 to 6.9 (inclusive), i.e., pH 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.43, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. In some embodiments, the pH of the finished / cosmetic composition comprising glyceryl caprylate / caprate ranges from pH 6.0 to 6.5 (inclusive), i.e., pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

[0321] In some embodiments, the arginine component of the cosmetic composition stabilization system includes a solvent. Examples of solvents include water, low molecular weight alcohols, e.g., C 1~6 Branched or straight chain alcohols, such as methanol, ethanol and isopropanol, low molecular weight ketones, such as C 1~6 Branched or straight chain ketones, such as acetone, aromatic compounds, and low molecular weight alkanes, such as C 1~10 Branched or straight chain alkanes may be mentioned.

[0322] In some embodiments, the arginine component comprises a polar solvent. Exemplary polar solvents include water, alcohol (such as ethanol, propyl alcohol, isopropyl alcohol, hexanol, benzyl alcohol, polyhydric alcohols, etc.), polyols (such as propylene glycol, polypropylene glycol, butylene glycol, hexylene glycol, polyethylene glycol, etc.), sugar alcohols (such as malitol, sorbitol), glycerin, panthenol dissolved in glycerin, fragrance oils, and mixtures thereof. Mixtures of these solvents can also be used. In some embodiments, the solvent is water.

[0323] In some embodiments, the solvent may be used in any suitable amount. For example, the solvent may be present in about 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, and about 95.0% of the total weight of the arginine component. Exemplary amounts include 10.0% to about 70.0% by weight (inclusive) based on the total weight of the arginine component, about 20.0% to about 60.0% by weight (inclusive) based on the total weight of the arginine component, and about 30.0% to about 50.0% by weight (inclusive) of the total weight of the arginine component.

[0324] In some embodiments, the pH range of the cosmetic or dermatological formulation stabilizing system is between pH 4.1 and 8.5 (inclusive), depending on the organic acid used, i.e., pH 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5.

[0325] In some embodiments, the cosmetic composition stabilizing system comprising the arginine component of the present invention may be present as at least about 0.001 wt.%, at least 0.005 wt.%, at least 0.01 wt.%, at least 0.05 wt.%, at least 0.10 wt.%, at least 0.50 wt.%, at least 1 wt.%, at least 2 wt.%, at least 3 wt.%, at least 4 wt.%, at least 5 wt.%, at least 6 wt.%, at least 7 wt.%, at least 8 wt.%, at least 9 wt.%, at least 10 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 55 wt.%, at least 60 wt.%, at least 65 wt.%, at least 70 wt.%, at least 75 wt.%, at least 80 wt.%, at least 85 wt.%, at least 90 wt.%, at least 95 wt.% of the total weight of the formulation.

[0326] In some embodiments, the cosmetic composition stabilizing system including the arginine component of the present invention may be present at about 1.0% to about 100.0%, about 10.0% to 70.0%, or about 20.0% to 50.0% by weight based on the total weight of the formulation. In some embodiments, the arginine component of the present invention may be present at about 0.001% to 10.0%, about 0.1% to 5.0%, or about 1.0% to 3.0% by weight based on the total weight of the formulation.

[0327] In some embodiments, the cosmetic stabilizing system comprises arginine levulinate in the range of about 0.25% to about 1.00% by weight (inclusive), i.e. 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.00% by weight of the composition, and arginine anisate in the range of about 0.05% to about 0.50% by weight (inclusive), i.e. 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% by weight of the composition.

[0328] In some embodiments, the pH of the cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid ranges from about pH 4.0 to about pH 6.0, inclusive, i.e., about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, or about pH 6.0.

[0329] The term "emulsion" as used herein refers to a two-phase system prepared by combining two immiscible liquid carriers, one of which is distributed uniformly throughout the other and consists of globules with diameters equal to or greater than that of the largest colloidal particle. The globule size is important and must be such that the system achieves maximum stability. Separation of the two phases occurs unless a third substance, usually an emulsifier, is incorporated.

[0330] An emulsifying agent (emulsifier) ​​is a compound or substance that acts as a stabilizer of an emulsion, preventing normally immiscible liquids from separating by increasing the dynamic stability of the mixture. The chemical structures of many of these agents have both hydrophilic and lipophilic portions. All emulsifiers concentrate and adsorb at the oil:water interface to provide a protective barrier around the dispersed droplets. In addition to this protective barrier, emulsifiers stabilize emulsions by lowering the interfacial tension of the system. Some agents increase stability by imparting an electric charge to the droplet surface, thus reducing the physical contact between the droplets and decreasing the possibility of bonding.

[0331] Emulsifiers can be classified according to: 1) chemical structure; or 2) mechanism of action. The classes according to chemical structure are synthetic, natural, finely dispersed solids, and adjuvants. The classes according to mechanism of action are monomolecular, polymolecular, and solid particle films. [https: / / pharmlabs.unc.edu / labs / emulsions / prep.htm, visited 10.16.20]

[0332] Exemplary synthetic emulsifiers include cationic, e.g., benzalkonium chloride, benzethonium chloride, anionic, e.g., alkali soaps (sodium or potassium oleate), amine soaps (triethanolamine stearate), detergents (sodium lauryl sulfate, dioctyl sodium sulfosuccinate, sodium docusate), and nonionic, e.g., sorbitan esters (Spans®), polyoxyethylene derivatives of sorbitan esters (Tweens®), or glyceryl esters. Cationic and anionic surfactants are generally limited to topical, o / w emulsion use. [Ibid.]

[0333] Various emulsifiers are natural products derived from plants or animal tissues. They form hydrated, freeze-dried colloids (called hydrocolloids) that form multilayers around the emulsion droplets. Hydrocolloid-type emulsifiers exert a protective colloid effect that has little or no effect on the interfacial tension, but reduces the likelihood of coalescence by providing a protective sheath around the droplets, imparting an electric charge to the dispersed droplets (so that they repel each other), and swelling to increase the viscosity of the system (so that the droplets are less likely to merge). Examples of hydrocolloid emulsifiers include, but are not limited to, plant derivatives such as acacia, tragacanth, agar, pectin, carrageenan, lecithin, animal derivatives such as gelatin, lanolin, cholesterol, semi-synthetic agents such as methylcellulose, carboxymethylcellulose, and synthetic agents such as Carbopols®. Animal derivatives generally form w / o emulsions. Lecithin and cholesterol form monolayers around the emulsion droplets instead of the typical multilayers. Cholesterol is the main component of wool alcohol, which gives lanolin the ability to absorb water and form emulsions. Lecithin (a phospholipid derived from egg yolk) produces o / w emulsions due to its strong hydrophilic properties. [Ibid.]

[0334] Finely divided or finely dispersed solid particle emulsifiers form a particle layer around the dispersed agglomerates. Most swell in the dispersion medium, increasing the viscosity and reducing the interactions between the dispersed droplets. Most commonly, they support the formation of o / w emulsions, although some may support w / o emulsions. Examples include bentonite, begum, hectorite, magnesium hydroxide, aluminum hydroxide, and magnesium trisilicate. [Ibid.]

[0335] The hydrophilic-lipophilic balance (HLB) system is used to describe the properties of surfactants, a class of emulsifiers, that reduce surface tension between liquids or between solids and liquids. It is an arbitrary scale in which HLB values ​​are experimentally determined and assigned. A low HLB value means that the number of hydrophilic groups on the surfactant is low, making it more lipophilic (oil-soluble) than hydrophilic (water-soluble). Conversely, a high HLB value means that the surfactant has a large number of hydrophilic groups, making it more hydrophilic (water-soluble) than oil-soluble. An HLB value of 10 or greater means that the agent is primarily hydrophilic.

[0336] Emulsifiers (HLB 3-6 (w / o) and 8-18 (o / w) are surfactants that reduce the interfacial tension between oil and water, thereby minimizing the surface energy through the formation of globules, examples include glyceryl monostearate, methylcellulose, sodium lauryl sulfate, sodium oleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, tragacanth, triethanolamine oleate, polyoxyethylene sorbitan monolaurate, poloxamer (Pluronic F-68)).

[0337] In some embodiments, the cosmetic composition of the present invention may contain a viscosity enhancing agent or thickening agent. A viscosity enhancing agent is an agent that thickens, gels, or hardens a composition. In some embodiments, the viscosity enhancing agent is derived from a plant extract. Exemplary viscosity enhancing agents include acacia, agar, algin, alginic acid, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluroinic acid, silicic acid, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, carboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and / or mixtures thereof.

[0338] In some embodiments, the emulsifier is Ecogel™, a commercially available phospholipid-based gelling agent with emulsifying properties that includes lysolecithin, sclerotium gum, xanthan gum, and pullulan. [Bay House Ingredients, Milton Keynes, England]. Ecogel™ is stable over a wide pH range (pH 2.0-10.0) and can be used as 0.25-1.0% by weight of the cosmetic formulation. In some embodiments, the cosmetic composition formulated for topical application is a slightly viscous, non-occlusive aqueous liquid that includes a cosmetic composition stabilizing system that includes arginine, p-anisic acid, and levulinic acid. In some embodiments, the pH of the finished product ranges from about 4.0 to about 5.0 (inclusive), i.e., 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some embodiments, the viscosity of the composition is in the range of 5000 to 7500 centipoise, i.e., about 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, or 7500 centipoise.

[0339] Exemplary formulation components are shown in Table 2. [Table 4]

[0340] method According to another aspect, the described invention provides a method for promoting / maintaining perianal tissue vitality and perianal tissue health in a subject, comprising topically administering a cosmetic composition comprising an aqueous gel component, a botanical ingredient component, and a cosmetic composition stabilizing system, wherein the cosmetic composition has a pH in the range of 4.0-5.0 inclusive, i.e., 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some embodiments, the subject is a female subject. In some embodiments, the cosmetic composition reduces one or more skin symptom(s) of hemorrhoidal disease. In some embodiments, the hemorrhoidal disease comprises external hemorrhoids. In some embodiments, the skin symptoms of hemorrhoidal disease include, but are not limited to, one or more of pain or itching. In some embodiments, the cosmetic composition may have one or more of the following cosmetic benefits: reducing symptoms and signs of trauma, injury, or damage (e.g., dryness, burning, irritation, discomfort, or pain); improving healing and rejuvenation of injured tissue (e.g., improving tissue strength). In some embodiments, the cosmetic composition reduces pain, itching, or both of external hemorrhoidal tissue. In some such embodiments, the cosmetic composition promotes wound healing of inflamed external hemorrhoidal tissue. In some embodiments, the described soothing compositions may restore healthy tissue properties through healing, rejuvenation, or both.

[0341] According to another aspect, the described invention provides a method for promoting / maintaining vaginal-vulvar tissue vitality and vaginal-vulvar tissue health in a female subject, comprising topically administering a cosmetic composition comprising an aqueous gel component, a botanical source component, and a cosmetic composition stabilizing system, wherein the cosmetic composition has a pH in the range of 4.0 to 5.0 (inclusive), i.e., 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0342] In some embodiments, the female subject is a subject who is susceptible to or has experienced urogenital symptoms of trauma, injury or injury. In some embodiments, the female subject is a menopausal subject. In some embodiments, the female subject is a diabetic subject. In some embodiments, the female subject is a subject who will undergo, is undergoing, or has undergone treatment, including radiation therapy, for treating gynecological cancer, such as endometrial cancer, cervical cancer, ovarian cancer, vulvar cancer. In some embodiments, the female subject is a breast cancer survivor.

[0343] In some embodiments, parameters of vaginal / vulvar tissue vitality include, but are not limited to, increased tissue strength, proper vaginal pH, decreased susceptibility to trauma / mechanical injury, reduced inflammation, reduced itching, and improved tissue elasticity.

[0344] In some embodiments, the cosmetic composition may have one or more of the following cosmetic benefits: reducing symptoms and signs of trauma, injury or damage (e.g., dryness, burning, irritation, discomfort or pain), improving healing and rejuvenation of injured tissue (e.g., improving tissue strength), reducing clinical signs of dryness and inadequate hydration (e.g., loss of elasticity, inflammation), or regulating vaginal pH, inflammation, or itching. In some embodiments, the cosmetic condition may create an environment that facilitates healing and rejuvenation of injured tissue. In some embodiments, the soothing compositions described may restore healthy tissue properties through healing, rejuvenation, or both.

[0345] Water-based gel component background Hyaluronic acid (HA) or hyaluronan is a member of a large family of glycosaminoglycans (GAGs) that are major components of the extracellular matrix. HA molecules consist of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-glycosidic bonds. [ka] This simple molecular unit has a molecular weight of 5 × 106 It forms long linear polymers up to 10 kDa. Long hyaluronan polymers have the ability to bind large amounts of water.

[0346] In its native form as a very long polymer, hyaluronan is known as high molecular weight (HMW) hyaluronan. Under certain conditions, it can be degraded into smaller fragments called low molecular weight HA. [Litwiniuk, M. et al. Wounds (2016) 28(3): 78-88, cited in Aya, KL & Stern, R. Wound Repair Regen. (2014) 22(5): 579-93]. In somatic tissues, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2) are responsible for HA degradation. First, Hyal-2, a plasma membrane-like enzyme, degrades HA into fragments of molecules8 up to 20 kDa. These HA molecules are subsequently endocytosed and delivered to lysosomes, where further digestion is carried out by Hyal-1. [Ibid. citing Stern, R. & Jedrzejas, M. Chem. Rev. (2006) 106(30:818-39). In damaged tissue, free radicals can also break down HA polymers into smaller fragments. [Ibid. citing Longacre, MT et al. Ann. Surg. (1991) 213(4):292-96].

[0347] It is well documented that HMWHA exhibits anti-inflammatory and immunosuppressive properties, whereas LMWHA degradation products of HA can induce inflammation. [Ibid. citing Prevo, R. et al. J. Biol. Chem. (2001) 276(22):19420-30]. Small hyaluronic acid fragments have been shown to increase the expression and protein production of several cytokines, such as MMP-12, plasminogen activator inhibitor-1 [Ibid., Horton, MR et al. Am. J. Physiol. Lung Cell Mol. Physiol. (2000) 279(4):L707-15, citing Horton, MR, et al. J Immunol. (1999) 162(7):4171-76], macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant 1, keratinocyte chemoattractant, interleukin-8 (IL-8), and IL-12 by macrophages. [Ibid., citing Horton, MR, et al. J. Immunol. (1998) 160(6):3023-30; Hodge-DuFour, J. et al. J. Immunol. (1997) 159(5):2492-2500; McKee, CM et al. J. Clin. Invest. (1996) 98(10):1403-13]. CD44 is the main receptor for hyaluronan, but other receptors, such as TLRs, are also involved in HA signaling. [Ibid., citing Teder, P. et al. Science (2002) 296(5565):155-58]. LMWHA can bind to TLR receptors and initiate a signaling cascade that leads to the production of proinflammatory cytokines and chemokines in various cell types in vitro. [Ibid. citing Litwiniuk, M. et al. Cent. Eur. J. Immunol. (2009) 34(4):247-51]. In immune cells from injured tissue, TLR2 and TLR4 activation by LMWHA has been shown to lead to the initiation of MyD88-dependent NFκB signaling cascade and pro-inflammatory cytokine gene expression.[Ibid., citing Jiang, D. et al. Nat. Med. (2005) 11(11): 1173-79, Suga, H. et al. J. Dermatol. Sci. (2014) 73(2): 117-24]. Induction of LMWHA by TLR-associated myeloid differentiation primary response gene 88 (MyD88) / NFκB signaling was also confirmed in breast tumor cells. Small HA fragments were shown to stimulate CD44 association with TLR2, TLR4 and MyD88, leading to NF-κB-specific transcriptional activation and expression of inflammatory cytokines Il-1β and IL-8 in human breast cell lines. Taken together, these reports suggest that LMWHA induces inflammation through activation of TLR receptors and initiation of MyD8 / NFκB signaling, which leads to the production of inflammatory cytokines and chemokines.

[0348] Under physiological conditions, activation of immune system cells is important for proper wound healing. In acute wounds, small hyaluronan fragments deposited at the site of injury activate the immune system to manage the disruption of tissue integrity, whereas in chronic wounds, a constant excessive inflammatory response has been shown to actually impede the healing of the wound. [Ibid.]

[0349] LMWHA, which has strong antioxidant properties, exhibits protective effects against ROS, inhibits lipid peroxidation, and scavenges free radicals both in vitro and in vivo. [Ibid., citing Ke, C. et al. Food Chem. Toxicol. (2011) 49(10):2670-75].

[0350] The anti-inflammatory potential of HMWHA is well documented in osteoarthritis. HA is a fundamental component of normal synovial fluid, and because HA concentrations are reduced in osteoarthritis-affected joints, intra-articular injections of HMWHA have been used to treat osteoarthritis. For example, HMWHA was able to inhibit IL-1β expression in synovial cells in a rabbit model of osteoarthritis. [Ibid., citing Miki, Y. et al. Inflamm. Res. (2010) 59(6):471-77]. IL-1-dependent expression of MMP-1 and MMP-3 was reduced in human synovial cells by HMWHA treatment. A large study analyzed the effect of HMWHA on gene expression of various inflammatory cytokines by human fibroblast-like synoviocytes (FLS) in patients with early osteoarthritis. [Ibid. citing Wang, CT, et al. osteoarthritis Cartilage (2006) 14(12):1237-47] They reported downregulation of IL-8 and iNOS gene expression in unstimulated FLS and aggrecanase-2, as well as tumor necrosis factor alpha (TNFα) gene expression in IL-1 stimulated FLS. Blocking the CD44 receptor with anti-CD44 antibodies inhibited the downregulatory effect of HMWHA on gene expression. [Ibid. citing Bourguignon, LY et al. Cytoskeleton (Hoboken) (2011) 68(12):671-93].

[0351] The exact mechanism by which HMWHA interacts with TLR receptors, leading to inhibition of the inflammatory cascade, is unknown. HWHA could significantly reduce the expression of TLR4, TLR2, MyD88, and NF-κB in synovial cells in a mouse model of osteoarthritis. [Ibid., citing Campo, GM et al. Biochim. Biophys. Acta (2011) 1812(9):1170-81]. They also observed a decrease in the mRNA expression and protein production of TNFα, IL-1β, IL-17, MMP-13, and inducible nitrous oxide synthase genes in arthritic mice treated with HMWHA. [Ibid., citing Campo, GM et al. Biochim. Biophys. Acta (2011) 1812(9):1170-81], but only when HWHA was administered in the early inflammatory phase of osteoarthritis.

[0352] Examples of the antioxidant effects of HMWHA include the reduction of UVB-induced apoptosis and EDTA-induced oxidative damage to DNA [ibid., Bourguignon, LY et al. J. Biol Chem. (1997) 272(44): 27913-27918, citing Pauloin, T. et al., Mol. Vis. (2009) 15: 577-83], and the reduction of apoptosis and oxidative stress caused by benzalkonium chloride and sodium lauryl sulfate detergents, which are widely used in pharmaceutical formulations [ibid., Pauloin, T. et al. Cornea (2009) 28(9): 1032-41, citing Pauloin, T. et al. Eur. J. Pharm. Sci. (20008) 34(4-5): 263-73]. The mechanism by which HMWHA reduces oxidative stress is not yet understood.

[0353] Many studies have shown that HA signaling plays a role in regulating angiogenesis, mainly by influencing endothelial cell behavior. Both HMWHA and LMWHA are potent regulators of angiogenesis. LMWHA stimulates vascular EC proliferation, migration, and tubule formation in vitro, as well as in various in vivo models of angiogenesis, while HMWHA exhibits antiangiogenic properties by inhibiting EC proliferation motility and sprouting formation. [Ibid., citing Toole, BP. Nat. Rev. Cancer (2004): 4(7): 528-39]. The exact molecular mechanisms that determine the pro- or antiangiogenic effects of different HA forms have not been fully elucidated. A context-dependent response to different forms of HA and a possible role of the microenvironment in this process have been suggested. In wound closure assays, the addition of CXCL12 to the culture medium significantly increased cell migration and induced faster wound closure. This effect was statistically enhanced when cells were preincubated with HMWHA. [Ibid. citing Fuchs, K. et al. Cell Death Dis. (2013) 4:e819]. In vitro studies showed that CXCR4 activation by CXCL12 was significantly increased in HUVECs pretreated with HMWHA, whereas preincubation with LMWHA blocked CXCL12 signaling in these cells.

[0354] In some embodiments, the sodium hyaluronate product (e.g., HMWHA, LMWHA, or both) is produced by biofermentation in a Steptococcus equi sub. Zooepidemicus strain. In some embodiments, the sodium hyaluronate product(s) is obtained from a commercial source (e.g., Bloomage Biotechnology Corp., Ltd., Jinan, China). In some embodiments, the main components of the biofermentation medium include wheat peptone, yeast extract powder, and glucose.

[0355] In some embodiments, the molecular weight of the HMWHA is in the range of 1000 to 1800 kDa (inclusive), i.e., at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa. In some embodiments, the molecular weight of the LMWHA is less than 10 kDa, i.e., at least 0.1 kDa to 10 kDa, at least 0.5 kDa to 10 kDa, at least 1 kDa to 10 kDa, at least 2 kDa to 10 kDa, at least 3 kDa to 10 kDa, at least 4 kDa to 10 kDa, at least 5 kDa to 10 kDa, at least 6 kDa to 10 kDa, at least 7 kDa to 10 kDa, at least 8 kDa to 10 kDa, or at least 9 kDa to 10 kDa.

[0356] In some embodiments, the finished product comprises about 0.10 to about 0.50 wt% (inclusive) LMWHA, i.e., 0.10 wt%, 0.15 wt%, 0.20 wt%, 0.25 wt%, 0.30 wt%, 0.35 wt%, 0.40 wt%, 0.45 wt%, or 0.50 wt% LMWHA, and about 0.50 to about 1.50 wt% (inclusive) HMWHA, i.e., 0.50 wt%, 0.60 wt%, 0.70 wt%, 0.80 wt%, 0.90 wt%, 1.0 wt%, 1.2 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, or 1.5 wt% HMHWA.

[0357] In some embodiments, the ratio of HMWHA to LMWHA is 1:0.7 to 1:1 (inclusive), ie, 1:0.7, 0.8, 0.9 or 1.0.

[0358] plant raw material ingredients In some embodiments, the plant material component is a cannabinoid. In some embodiments, the cannabinoid is derived from hemp. In some embodiments, the cannabinoid is not full spectrum CBD, i.e., does not contain all the cannabinoids found in the natural cannabis plant. In some embodiments, the cannabinoid is non-psychoactive. In some embodiments, the cannabinoid is decarboxylated CBD.

[0359] In some embodiments, the isolate comprises about 20% decarboxylated CBD. In some embodiments, the isolate comprising about 20% decarboxylated CBD is in the form of a THC-free nano-infused water-soluble powder. In some embodiments, the isolate comprising about 20% decarboxylated CBD is obtained from a commercial source (e.g., Global Cannabinoids, Las Vegas, NV). In some embodiments, the isolate comprising about 20% CBD comprises less than 1% cannabidivarin (CBDV), which has a molecular structure similar to CBD, but instead of having a pentyl chain, it has a propyl chain. In some embodiments, the decarboxylation is performed by heating to not more than 300°F, e.g., 200°F (93°C) for 75 minutes, 225°F (107°C) for 50 minutes, or 250°F (121°C) for 30 minutes.

[0360] An exemplary analysis of THC-free nano-infused water soluble cannabinoid powder is shown below (LOQ = Limit of Quantitation). Results reported are based on sample weight at the appropriate moisture content for that sample. Unless otherwise noted, all quality control specimens perform within specifications established by the laboratory. [Table 5]

[0361] In some embodiments, total CBD can be expressed as the formula CBDa*0.877+CBD and total THC can be expressed as THCa*0.877+Δ9-THC+Δ8THC. In some embodiments, the powder contains one or more inactive ingredients, such as tapioca maltodextrin.

[0362] In some embodiments, the finished product comprises between 1.0% and about 5.0% by weight (inclusive), i.e., at least 1.0%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2.0%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3.0%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4.0%, at least 4.25%, at least 4.5%, at least 4.75%, or at least 5% by weight of the 20% CBD-THC free nano-infused water soluble powder.

[0363] Cosmetic composition stabilization system In some embodiments, the cosmetic composition stabilizing system comprises an effective amount of an arginine component. In some embodiments, the arginine component comprises i) arginine, or a conjugate, or an analog thereof, ii) an organic acid, a conjugate, or an analog thereof, and iii) a solvent. As used herein, the phrase "arginine, a conjugate, or an analog thereof" is referred to as an "arginine compound." As used herein, the phrase "organic acid, a conjugate, or a derivative thereof" is referred to as an "organic acid compound." In some embodiments, the arginine component comprises i) an arginine compound, ii) an organic acid compound, and iii) a solvent.

[0364] In some embodiments, the cosmetic composition stabilizing system has a preservative effect, a solubilizing effect, a stabilizing effect, a neutralizing or antimicrobial effect, a moisturizing and / or healing effect on the composition, or any combination thereof. For example, in some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition and / or a solubilizing effect on the active agent. According to some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing effect and / or a neutralizing effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, stabilizing, and / or a neutralizing, and / or antibacterial effect on the composition. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing effect on the active agent, and / or a stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing cosmetic / therapeutic effect. In some embodiments, the cosmetic composition stabilizing system has a preservative effect on the composition, and / or a solubilizing, stabilizing, neutralizing, and / or antibacterial effect on the composition, and / or a moisturizing, exfoliating, and / or healing cosmetic / therapeutic effect.

[0365] In some embodiments, the arginine compound is arginine, or its salt, conjugate, or analog.For example, arginine can be D, L-arginine, D-arginine, L-arginine, alkyl (ethyl, methyl, propyl, isopropyl, butyl, isobutyl, t-butyl) esters of arginine, and conjugates and analogs thereof.

[0366] In some embodiments, the arginine conjugate may be a conjugate acid or a conjugate base, for example, the arginine conjugate may be arginine, or an arginate salt.

[0367] In some embodiments, the arginine compound may be used in any suitable amount. For example, the arginine compound may be present in the arginine component at least about 1.0% by weight, at least about 5.0% by weight, at least 10.0% by weight, at least about 15.0% by weight, at least 20.0% by weight, at least 25.0% by weight, at least 30.0% by weight, at least 35.0% by weight, at least 40.0% by weight, at least 45.0% by weight, at least 50.0% by weight, at least 55.0% by weight, at least 60.0% by weight, at least 65.0% by weight, at least 70.0% by weight, at least 75.0% by weight, at least 80.0% by weight, at least 85.0% by weight, at least 90.0% by weight, and about 95.0% by weight of the total weight of the arginine component. Exemplary amounts include 10.0% to about 70.0% by weight (inclusive) based on the total weight of the arginine component, about 20% to about 60% by weight (inclusive) based on the total weight of the arginine component, and about 30% to about 50% by weight (inclusive) of the total weight of the arginine component.

[0368] In some embodiments, the organic acid compound can be an organic acid, its conjugate, or its analog. In some embodiments, the organic acid can be any suitable organic acid. For example, the organic acid can be substituted and unsubstituted aliphatic (saturated and unsaturated) and aromatic acids. The organic acid can have one or more functional groups as a substituent, such as alkyl, alkenyl, alkynyl, halogen, hydroxy, carbonyl, carboxylic acid, aldehyde, ester, amide, carbonate, carbamate, ether, amino, cyano, isocyano, oxy, oxo, thia, aza, azide, imine, nitro, nitrosooxy, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfhydryl, sulfonyl, phosphino, etc., where each of the alkyl, alkenyl, alkynyl, and amino groups can be optionally substituted with one or more of the aforementioned functional groups. In some embodiments, some functional groups, such as hydroxy, impart or enhance properties to the acid that are suitable for the present composition, such as hygroscopic properties. In some embodiments, the organic acid comprises multiple carboxylic acid groups.

[0369] In some embodiments, the organic acid is a hydroxy acid, in some embodiments, the hydroxy acid is an alpha-hydroxy acid (AHA), beta-hydroxy acid (BHA), gamma-hydroxy acid (GBA), omega-hydroxy acid, monohydroxy acid (MHA), polyhydroxy acid (PHA) / polycarboxyhydroxy acid (PCHA), aliphatic hydroxy acid (AlHA), aromatic hydroxy acid (ArHA), aliphatic hydroxy acid (AAHA), hydroxy fatty acid, etc.

[0370] In some embodiments, the hydroxy acid is an alpha-hydroxy acid (AHA). The alpha (α) carbon in an organic molecule refers to the first carbon atom that is bonded to a functional group such as a carbonyl. In some embodiments, the AHA is an alkyl AHA, an aryl alkyl AHA, or a polycarboxyl AHA.

[0371] In some embodiments, the organic acid component comprises anisic acid, also known as 2-methoxybenzoic acid, o-anisic acid, 579-75-9, o-methoxybenzoic acid, 2-anisic acid, O-methylsalicylic acid, benzoic acid, 2-methoxy-, salicylic acid methyl ether, 2-methoxybenzoic acid, 529-75-9, molecular formula CHO.

[0372] In some embodiments, the organic acid component comprises levulinic acid, also known as 4-oxopentanoic acid; 123-76-2; laevulinic acid; pentanoic acid, 4-oxo-4-oxovaleric acid; levulinic acid; 3-acetylpropionic acid; 4-ketovaleric acid; LEVA; molecular formula C5H8O3.

[0373] In some embodiments, the organic acid component comprises mandelic acid, also known as 17199-29-0, (S)-(+)-mandelic acid, (S)-mandelic acid, (S)-2-hydroxy-2-phenylacetic acid, L-mandelic acid, L-(+)-mandelic acid, S-(+)-mandelic acid, (2S)-2-hydroxy-2-phenylacetic acid, UNII-L0UMW58G3T, L(+)-mandelic acid, molecular formula CHO.

[0374] In some embodiments, the organic acid component comprises salicylic acid, also known as 2-hydroxybenzoic acid, 69-72-7, o-hydroxybenzoic acid, 2-carboxyphenol, o-carboxyphenol, Rutranex, Salonil, Retarder W, Keralyt, with the molecular formula C7H6O3 or HOC6H4COOH.

[0375] In some embodiments, the organic acid component comprises sorbic acid, also known as 110-44-1, 2,4-hexadienoic acid, (2E,4E)-hexa-2,4-dienoic acid, 2E,4E-hexadienoic acid, Panosorb, Sorbistat, 2-propenylacrylic acid, trans,trans-sorbic acid, hexadienoic acid, molecular formula CHO or CHCH=CHCH=CHCOOH.

[0376] In some embodiments, the organic acid component comprises benzoic acid, also known as 65-85-0, dolacyl acid, benzenecarboxylic acid, carboxybenzene, benzeneformic acid, phenylformic acid, benzenemethanoic acid, phenylcarboxylic acid, Retardex, with the molecular formula C7H6O2 or C6H5COOH.

[0377] In some embodiments, the organic acid component includes ferulic acid, also known as trans-4-hydroxy-3-methoxycinnamic acid, and trans-ferulic acid, with the molecular formula HOC6H3(OCH3)CH=CHCO2H.

[0378] In some embodiments, the organic acid component includes 3,5-dimethoxy-4-hydroxybenzoic acid, 4-hydroxy-3,5-dimethoxy-benzoic acid, and syringic acid, also known as gallic acid 3,5-dimethyl ether, molecular formula HOC6H2(OCH3)2CO2H.

[0379] In some embodiments, the organic acid component is anisic acid, levulinic acid, mandelic acid, salicylic acid, sorbic acid, benzoic acid, ferulic acid, and syringic acid, e.g., anisic acid and levulinic acid, anisic acid and mandelic acid, anisic acid and salicylic acid, anisic acid and sorbic acid, anisic acid and benzoic acid, anisic acid and ferulic acid, anisic acid and syringic acid, levulinic acid and mandelic acid, levulinic acid and salicylic acid, levulinic acid and sorbic acid, levulinic acid and benzoic acid, levulinic acid and ferulic acid, levulinic acid and syringic acid, mandelic acid and salicylic acid, mandelic acid and sorbic acid, mandelic acid and benzoic acid, mandelic acid and ferulic acid, mandelic acid and syringic acid, salicylic acid acid) and two organic acids selected from the group consisting of sorbic acid, salicylic acid and benzoic acid, salicylic acid and ferulic acid, salicylic acid and syringeic acid, sorbic acid and benzoic acid, sorbic acid and ferulic acid, sorbic acid and syringeic acid, benzoic acid and ferulic acid, benzoic acid and syringeic acid, ferulic acid and syringeic acid.

[0380] In some embodiments, organic acids, such as a combination of the two exemplified above (e.g., levulinic acid and anisic acid), when used with glyceryl caprylate / caprate, form a broad-spectrum preservative that is effective against microbial contaminants, such as bacteria, yeasts, and molds. Without being limited to any particular theory, a stabilization system that includes glyceryl caprylate / caprate allows greater penetration of both acids into the cell walls of the target organism, which means that the overall concentration of acid in the formulation is lower, and the concentration of free acid required for full-spectrum preservation of the product is reduced. This, in turn, increases the effectiveness in finished formulations that are difficult at physiological pH, and reduces the need for additional ingredients in the finished formulation without drastic adjustment of the final pH of the finished formulation.

[0381] In some embodiments, organic acid compounds include any derivatives of the organic acid, for example derivatives of organic acids that revert to their acid form when contacted with water, including, but not limited to, readily hydrolyzable anhydride, mixed anhydride, and ester derivatives of organic acids.

[0382] In some embodiments, the organic acid compound may be used in any suitable amount. For example, the organic acid compound may be present in the arginine component as about 1.0 wt%, 5.0 wt%, 10.0 wt%, 15.0 wt%, 20.0 wt%, 25.0 wt%, 30.0 wt%, 35.0 wt%, 40.0 wt%, 45.0 wt%, 50.0 wt%, 55.0 wt%, 60.0 wt%, 65.0 wt%, 70.0 wt%, 75.0 wt%, 80.0 wt%, 85.0 wt%, 90.0 wt%, and about 95.0 wt% of the total weight of the arginine component. Exemplary amounts include about 10.0 wt% to about 70.0 wt% based on the total weight of the arginine component, about 20 wt% to about 60 wt% based on the total weight of the arginine component, and about 30 wt% to about 50 wt% of the total weight of the arginine component.

[0383] In some such embodiments, the pH is high enough to keep the acid in solution and low enough to keep glyceryl caprylate / caprate from hydrolysis. For example, the pH of the raw cosmetic composition stabilizing system in water containing glyceryl caprylate / caprate can range from pH 4.1 to 6.9 (inclusive), i.e., pH 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.43, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. In some embodiments, the pH of the finished / cosmetic composition comprising glyceryl caprylate / caprate ranges from pH 6.0 to 6.5 (inclusive), i.e., pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

[0384] In some embodiments, the arginine component of the cosmetic composition stabilization system includes a solvent. Examples of solvents include water, low molecular weight alcohols, e.g., C 1~6 Branched or straight chain alcohols, such as methanol, ethanol and isopropanol, low molecular weight ketones, such as C 1~6 Branched or straight chain ketones, such as acetone, aromatic compounds, and low molecular weight alkanes, such as C 1~10 Branched or straight chain alkanes may be mentioned.

[0385] In some embodiments, the arginine component comprises a polar solvent. Exemplary polar solvents include water, alcohol (such as ethanol, propyl alcohol, isopropyl alcohol, hexanol, benzyl alcohol, polyhydric alcohols, etc.), polyols (such as propylene glycol, polypropylene glycol, butylene glycol, hexylene glycol, polyethylene glycol, etc.), sugar alcohols (such as malitol, sorbitol), glycerin, panthenol dissolved in glycerin, fragrance oils, and mixtures thereof. Mixtures of these solvents can also be used. In some embodiments, the solvent is water.

[0386] In some embodiments, the solvent may be used in any suitable amount. For example, the solvent may be present in about 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, and about 95.0% of the total weight of the arginine component. Exemplary amounts include 10.0% to about 70.0% by weight (inclusive) based on the total weight of the arginine component, about 20.0% to about 60.0% by weight (inclusive) based on the total weight of the arginine component, and about 30.0% to about 50.0% by weight (inclusive) of the total weight of the arginine component.

[0387] In some embodiments, the pH range of the cosmetic or dermatological formulation stabilizing system is between pH 4.1 and 8.5 (inclusive), depending on the organic acid used, i.e., pH 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5.

[0388] In some embodiments, the cosmetic composition stabilizing system comprising the arginine component of the present invention may be present as at least about 0.001 wt.%, at least 0.005 wt.%, at least 0.01 wt.%, at least 0.05 wt.%, at least 0.10 wt.%, at least 0.50 wt.%, at least 1 wt.%, at least 2 wt.%, at least 3 wt.%, at least 4 wt.%, at least 5 wt.%, at least 6 wt.%, at least 7 wt.%, at least 8 wt.%, at least 9 wt.%, at least 10 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.%, at least 55 wt.%, at least 60 wt.%, at least 65 wt.%, at least 70 wt.%, at least 75 wt.%, at least 80 wt.%, at least 85 wt.%, at least 90 wt.%, at least 95 wt.% of the total weight of the formulation.

[0389] In some embodiments, the cosmetic composition stabilizing system including the arginine component of the present invention may be present at about 1.0% to about 100.0%, about 10.0% to 70.0%, or about 20.0% to 50.0% by weight based on the total weight of the formulation. In some embodiments, the arginine component of the present invention may be present at about 0.001% to 10.0%, about 0.1% to 5.0%, or about 1.0% to 3.0% by weight based on the total weight of the formulation.

[0390] In some embodiments, the cosmetic stabilizing system comprises arginine levulinate in the range of about 0.25 to about 1.00% by weight (inclusive) of the composition, i.e., 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1.00%, and arginine anisate in the range of about 0.05% to about 0.50% by weight of the composition, i.e., 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%.

[0391] In some embodiments, the pH of the cosmetic composition stabilization system comprising arginine, p-anisic acid, and levulinic acid ranges from pH 7.0 to pH 7.9 inclusive, i.e., pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, or pH 7.9.

[0392] The term "emulsion" as used herein refers to a two-phase system prepared by combining two immiscible liquid carriers, one of which is distributed uniformly throughout the other and consists of globules with diameters equal to or greater than that of the largest colloidal particle. The globule size is important and must be such that the system achieves maximum stability. Separation of the two phases occurs unless a third substance, usually an emulsifier, is incorporated.

[0393] An emulsifying agent (emulsifier) ​​is a compound or substance that acts as a stabilizer of an emulsion, preventing normally immiscible liquids from separating by increasing the dynamic stability of the mixture. The chemical structures of many of these agents have both hydrophilic and lipophilic portions. All emulsifiers concentrate and adsorb at the oil:water interface to provide a protective barrier around the dispersed droplets. In addition to this protective barrier, emulsifiers stabilize emulsions by lowering the interfacial tension of the system. Some agents increase stability by imparting an electric charge to the droplet surface, thus reducing the physical contact between the droplets and decreasing the possibility of bonding.

[0394] Emulsifiers can be classified according to: 1) chemical structure; or 2) mechanism of action. The classes according to chemical structure are synthetic, natural, finely dispersed solids, and adjuvants. The classes according to mechanism of action are monomolecular, polymolecular, and solid particle films. [https: / / pharmlabs.unc.edu / labs / emulsions / prep.htm,visited 10.16.20]

[0395] Exemplary synthetic emulsifiers include cationic, e.g., benzalkonium chloride, benzethonium chloride, anionic, e.g., alkali soaps (sodium or potassium oleate), amine soaps (triethanolamine stearate), detergents (sodium lauryl sulfate, dioctyl sodium sulfosuccinate, sodium docusate), and nonionic, e.g., sorbitan esters (SPANS®), polyoxyethylene derivatives of sorbitan esters (TWEENS®), or glyceryl esters. Cationic and anionic surfactants are generally limited to topical, o / w emulsion use. [Ibid.]

[0396] Various emulsifiers are natural products derived from plants or animal tissues. They form hydrated, freeze-dried colloids (called hydrocolloids) that form multilayers around the emulsion droplets. Hydrocolloid-type emulsifiers exert a protective colloid effect that has little or no effect on the interfacial tension, but reduces the likelihood of coalescence by providing a protective sheath around the droplets, imparting an electric charge to the dispersed droplets (so that they repel each other), and swelling to increase the viscosity of the system (so that the droplets are less likely to merge). Examples of hydrocolloid emulsifiers include, but are not limited to, plant derivatives such as acacia, tragacanth, agar, pectin, carrageenan, lecithin, animal derivatives such as gelatin, lanolin, cholesterol, semi-synthetic agents such as methylcellulose, carboxymethylcellulose, and synthetic agents such as Carbopols®. Animal derivatives generally form w / o emulsions. Lecithin and cholesterol form monolayers around the emulsion droplets instead of the typical multilayers. Cholesterol is the main component of wool alcohol, which gives lanolin the ability to absorb water and form emulsions. Lecithin (a phospholipid derived from egg yolk) produces o / w emulsions due to its strong hydrophilic properties. [Ibid.]

[0397] Finely divided or finely dispersed solid particle emulsifiers form a particle layer around the dispersed agglomerates. Most swell in the dispersion medium, increasing the viscosity and reducing the interactions between the dispersed droplets. Most commonly, they support the formation of o / w emulsions, although some may support w / o emulsions. Examples include bentonite, begum, hectorite, magnesium hydroxide, aluminum hydroxide, and magnesium trisilicate. [Ibid.]

[0398] The hydrophilic-lipophilic balance (HLB) system is used to describe the properties of surfactants, a class of emulsifiers, that reduce surface tension between liquids or between solids and liquids. HLB values ​​are arbitrary scales that are experimentally determined and assigned. A low HLB value means that the number of hydrophilic groups on the surfactant is low, meaning that it is more lipophilic (oil-soluble) than hydrophilic (water-soluble). Conversely, a high HLB value means that the surfactant has a large number of hydrophilic groups, making it more hydrophilic (water-soluble) than oil-soluble. An HLB value of 10 or greater means that the agent is primarily hydrophilic.

[0399] Emulsifiers (HLB 3-6 (w / o) and 8-18 (o / w) are surfactants that reduce the interfacial tension between oil and water, thereby minimizing the surface energy through the formation of globules, examples include glyceryl monostearate, methylcellulose, sodium lauryl sulfate, sodium oleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, tragacanth, triethanolamine oleate, polyoxyethylene sorbitan monolaurate, poloxamer (Pluronic F-68)).

[0400] In some embodiments, the cosmetic composition of the present invention may contain a viscosity enhancing agent or thickening agent. A viscosity enhancing agent is an agent that thickens, gels, or hardens a composition. In some embodiments, the viscosity enhancing agent is derived from a plant extract. Exemplary viscosity enhancing agents include acacia, agar, algin, alginic acid, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluroinic acid, silicic acid, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, carboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and / or mixtures thereof.

[0401] In some embodiments, the emulsifier is Ecogel™, a commercially available phospholipid-based gelling agent with emulsifying properties that contains lysolecithin, sclerotium gum, xanthan gum, and pullulan. [Bay House Ingredients, Milton Keynes, England]. Ecogel™ is stable over a wide pH range (pH 2.0-10.0) and can be used as 0.25-1.0% by weight of the cosmetic formulation.

[0402] In some embodiments, a cosmetic composition formulated for topical application and comprising an aqueous gel component comprising LMWHA and HMWHA, and a botanical component comprising decarboxylated CBD present at a concentration of about 20% in the form of a THC-free nano-infused water-soluble powder is a slightly viscous, non-occlusive aqueous liquid comprising a cosmetic composition stabilizing system comprising arginine, p-anisic acid, levulinic acid, and lactic acid. In some embodiments, the pH of the finished product is about 4.5. In some embodiments, the viscosity is between 5000 and 7500 centipoise, i.e., about 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, or 7500 centipoise.

[0403] Exemplary formulation components are shown in Table 3. [Table 6]

[0404] When a range of values ​​is provided, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limit of that range, and at any other stated or intermediate value within that stated range, is encompassed within the invention to the tenth of the unit of the lower limit. The upper and lower limits of these smaller ranges, which may be independently included in the smaller ranges, are also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0405] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention, exemplary methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0406] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0407] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed. EXAMPLES

[0408] The following examples are put forward to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0409] Introduction As used herein, the term "skin" refers to the membranous protective covering of the body consisting of the epidermis and the corium (dermis).

[0410] Anatomy and Physiology of the Skin Skin is the largest organ in the body, consisting of several layers, and plays a key role in biological homeostasis. Reapproximation on the wound surface has long been the primary sign that the majority of wound healing is complete. Reclosure of this defect not only restores the protective functions of the skin, including protection from bacteria, toxins, and mechanical forces, but also provides a barrier to retain essential body fluids. The epidermis, composed of several layers starting from the stratum corneum, is the outermost layer of the skin. The innermost skin layer is the deep dermis. Skin has multiple functions, including thermoregulation, metabolic functions (vitamin D metabolism), and immune functions. Figure 1 shows a schematic diagram of the anatomy of the skin.

[0411] epidermis It is the function of the epidermis to close wounds quickly and efficiently. The epidermis provides a buffer zone against the environment; it provides protection from trauma, excludes toxins and microorganisms, provides a semipermeable membrane, and keeps vital body fluids within a protective envelope. Traditionally, the epidermis is divided into several layers, two of which represent the most physiologically important. The basal cell layer, or stratum germinativum, is important because it is the main source of regenerative cells. In the process of wound healing, this is most often the area that undergoes mitosis. The upper epidermis, including the stratum granulosum and stratum granulosum, is important because it is the area of ​​formation of normal epidermal barrier function.

[0412] When the epidermis is damaged, the body is subject to the entry of external agents and loss of fluids. Epidermal wounds heal primarily by cell migration. Clusters of epidermal cells migrate to the damaged area and cover the defect. These cells are phagocytic, clearing the surface of debris and plasma clots. Repair cells originate from local sources, primarily the dermal appendages, and from adjacent uninjured skin areas. Healing occurs rapidly, the skin is regenerated, and no scars are left. Blisters are an example of epidermal wounds. These can be small or larger blisters (blisters over 1 cm in diameter).

[0413] Stratum corneum and acid mantle The stratum corneum is an avascular multi-layered structure that acts as a barrier to the environment and prevents transepidermal water loss. Studies have shown that enzymatic activity is involved in the formation of an acid mantle in the stratum corneum. Together, the acid mantle and stratum corneum reduce the permeability of the skin to water and other polar compounds, indirectly protecting the skin from microbial invasion. Normal superficial skin pH is 4-6.5 in healthy individuals and varies with the area of ​​skin on the body. This low pH forms an acid mantle that enhances the skin barrier function. Damage to the stratum corneum increases the pH of the skin and therefore increases the susceptibility of the skin to bacterial skin infections.

[0414] Other layers of the epidermis Other layers of the epidermis below the stratum corneum include the stratum lucidum, stratum granulosum, stratum germinativum, and stratum basale. Each contains living cells with specialized functions (Figure 2). For example, melanin, produced by melanocytes in the epidermis, is responsible for the color of the skin. Langerhans cells are involved in immune processing.

[0415] skin appendages Skin appendages, including hair follicles, sebaceous and sweat glands, fingernails, and toenails, originate from the epidermis and project into the dermis, while sebaceous and sweat glands contribute epithelial cells for rapid re-epithelialization of wounds that do not penetrate the dermis (called partial wounds). Sebaceous glands are responsible for secretions that keep the skin smooth, soft, and supple. They are most abundant on the face and sparse on the palms and soles. Sweat gland secretions control the pH of the skin and prevent skin infections. Sweat glands, cutaneous blood vessels, and small muscles in the skin (which cause goosebumps) control the temperature of the body's surface. Nerve endings in the skin include receptors for pain, touch, heat, and cold. Loss of these nerve endings increases the risk of skin breakdown by reducing the tissue's resistance to external forces.

[0416] The basement membrane separates and connects the epidermis and dermis. When epithelial cells within the basement membrane divide, one cell remains and the other migrates through the granular layer to the superficial stratum corneum. At the surface, cells die and form keratin. The dried keratin on the surface is called scale. Hyperkeratosis (thick layer of keratin) is often seen on the heels and indicates loss of sebaceous and sweat gland function if the patient is diabetic. The basement membrane atrophies with aging, and separation between the basement membrane and the dermis is one of the causes of skin fissures in the elderly.

[0417] dermis The dermis, or true skin, is the vascular structure that supports and nourishes the epidermis. It also contains sensory nerve endings that transmit signals related to pain, pressure, heat, and cold. The dermis is divided into two layers. The superficial dermis is composed of extracellular matrix (collagen, elastin, and ground substance) and contains blood vessels, lymphatic vessels, epithelial cells, connective tissue, muscle, fat, and nerve tissue. The vascular supply of the dermis is responsible for nourishing the epidermis and regulating body temperature. Fibroblasts are responsible for producing the collagen and elastin components of skin that give it volume. Fibronectin and hyaluronic acid are secreted by fibroblasts.

[0418] The second layer, the deep dermis, lies above the subcutaneous fat and contains a larger network of blood vessels and collagen fibers to provide tensile strength. It is also composed of fibroelastic connective tissue, which is yellow in color and composed mainly of collagen. Fibroblasts are also present in this tissue layer. The well-vascularized dermis withstands pressure for a longer period of time than the subcutaneous tissue or muscle. Dermal collagen gives the skin its toughness. Dermal wounds, such as fissures or pustules, involve the epidermis, basement membrane, and dermis. Typically, skin injuries heal rapidly. Fissures in the dermis can exude serum, blood, or pus, leading to the formation of a blood clot or crust. Pustules are pus-filled vesicles that often represent infected hair follicles.

[0419] One non-limiting example of an effect on the skin surface is the formation of a film. The film formation can be protective (e.g., sunscreen) and / or occlusive (e.g., to provide a moisturizing effect by reducing water loss from the skin surface). One non-limiting example of an effect within the stratum corneum is skin moisturization, which can involve hydration of dry outer cells by a surface film or intercalation of water in lipid-rich intercellular lamina, and the stratum corneum can also function as a reservoir phase or depot where topically applied substances accumulate by partitioning into or binding to skin components.

[0420] Example 1. Safety Study, LMWHA (miniHA™) (1) Cytotoxicity test of miniHA (trademark) molecular weight 8,300 Da material:

[0421] Epithelial cell line: mouse fibroblast cell line L929 (Chinese Academy of Science Type Culture Collection).

[0422] LMWHA, molecular weight 8,300 Da, degraded by Bacillus hyaluronidase, provided by Bloomage Freda Biopharm Co., Ltd. ("mini-HA").

[0423] HA-Oligo (molecular weight 8000) produced by chemical decomposition.

[0424] method Two preparations of LMWHA were compared: LMWHA with a molecular weight of 8,300 Da enzymatically degraded by Bacillus hyaluronidase, and HA-Oligo (molecular weight 8,000) produced by chemical degradation.

[0425] Different concentrations of Mini-HA or HA-Oligo in solution were added to monolayer cultures of L929 fibroblasts in complete medium to achieve final concentrations of 0.25%, 0.5%, 1%, 2% and 3% (w / v). Cultures were incubated at 37°C in a humid atmosphere with 5% CO2. Test samples were as follows: (1) complete medium, (2) complete medium + miniHA™, (3) complete medium + HA-oligo.

[0426] Figures 3A-C show micrographs of L929 fibroblast cytotoxicity tests: Figure 3A, complete medium (control); Figure 3B, complete medium + miniHA™; Figure 3C, complete medium + HA-Oligo. No morphological cytotoxicity was observed.

[0427] A plot of the relative growth rate (RGR) versus the concentration of HA oligosaccharides (%, w / v) added to in vitro cultures of L929 fibroblasts is shown in Figure 4. The results show that there was no adverse effect on cell growth when less than 2% (w / v) of miniHA was added to the medium. Based on the US Pharmacopoeia cytotoxicity grade (toxicity = relative growth rate (RGR) < 50%), a concentration of 3% (w / v) of miniHA™ is considered non-toxic. In contrast, significant cytotoxicity was observed when 2% (w / v) of HA-Oligo was added to the medium. Such cytotoxicity is believed to be due to by-products of acid hydrolysis, such as furan-like and cyclopentanone derivatives. [See Smejkalova, D. et al. Carbohydrate Polymers (2012) 88:1425-34].

[0428] (2) Skin patch test of miniHA(trademark) 0.5 ml of 1% miniHA™ solution was administered twice daily for 7 days to the flexible forearms (5×5 cm) of 20 healthy volunteers. 2 ) and observed the skin reaction. The results are shown in Table 4. [Table 7]

[0429] Conclusion: Skin reactions to miniHA were negative in all volunteers.

[0430] Based on the results of the cytotoxicity test and the skin patch test, it can be concluded that miniHA™ is a safe cosmetic ingredient.

[0431] Example 2: Evaluation of the effect of LMWHA (miniHA™) and HMWHA on hydration / moisturization of human skin Skin hydration is primarily related to the skin barrier function and is important for maintaining a healthy skin barrier. The role of moisturizing skin care products is to act on the skin surface to physically limit water loss (occlusive effect) or at the cellular level.

[0432] Background. Dielectric constant, also called relative permittivity or specific inductive capacitance, is a property of an electrically insulating material (dielectric) that is equal to the ratio of the capacitance of a capacitor filled with a given material to the capacitance of an identical capacitor in a vacuum without the dielectric material. The insertion of a dielectric between the plates of a parallel plate capacitor always increases its capacitance, or ability to store opposite charges on each plate, compared to this ability when the plates are separated by a vacuum. The dielectric constant, represented by the Greek letter kappa, κ, is simply expressed as κ=C / C0, where C is the value of the capacitance of a capacitor filled with a given dielectric and C0 is the capacitance of the identical capacitor in a vacuum. The value of the static dielectric constant of any material is always greater than 1, which is its value for a vacuum. The values ​​of the dielectric constant at room temperature (25°C) are 1.00059 for air and 78.2 for water.

[0433] The electrical properties of the skin depend on the water content of the stratum corneum of the epidermis. Epidermal hydration can be assessed by measuring the electrical capacitance with the help of the stratum corneum moisture meter CM825. The principle of the method is based on the difference between the dielectric constant of water and other substances by measuring the capacitance of the dielectric. (Constantin,MM et al.“Skin hydration assessment through modern non-invasive bioengineering technologies.(2014)Maedica 9(10:33-38). Any change in the dielectric constant following a variation in the skin surface hydration results in a reduction in the calculated capacitance of the capacitor.

[0434] In general, the variation range of skin hydration values ​​is 0-130 arbitrary units (AU). Under standard working conditions (T°=20-22°C, humidity 40-60%), the variation of skin hydration values ​​of the anterior central forearm is as follows: below 30 AU - very dry, between 30-45 AU - dry, 45 AU - well hydrated.

[0435] The advantages of this method are the very short measurement time (1 s), the high reproducibility of the measurements and the absence of galvanic contact between the measurement area and the measuring device (the obtained results are not affected by ionic conductivity or polarization effects). In addition, the modern electronics of the probe allow temperature stability and eliminate interference of base capacitance and fluctuations in the power supply. Compared to other methods, preparations applied to the skin have little effect on the measurement, while being able to detect slight changes in the hydration level.

[0436] Methods. Thirty healthy volunteers, ranging in age from 20 to 50 years, were examined using a 4×4 cm smear on the left (control) and right (test sample) forearms. 2 ) to the sample (3.0±0.01mg / cm 2 The degree of skin hydration after topical application was determined at multiple time points, i.e., 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours, and compared to the control (before application).

[0437] Figure 5A shows the water retention capacity of miniHA™, molecular weight 8,300 Da, compared to a control. Figure 5B shows the water retention capacity of high molecular weight hyaluronic acid, molecular weight 1,170 kDa, compared to a control. The data show that miniHA™ has the same water retention capacity as the high molecular weight HAs tested.

[0438] 6 is a graph of stratum corneum moisture content (%) versus time for 0.1% miniHA, 0.2% miniHA, and 0.5% HA. As shown, the higher concentration of miniHA, i.e., 0.5% miniHA™, has better moisture retention capacity than either 0.1% or 0.2% miniHA.

[0439] Figure 7 is a graph of the stratum corneum moisture content (%) versus time for 0.2% HA (molecular weight 1,630,000 DA), 0.2% miniHA, and 0.1% HA + 0.1% miniHA. As shown, when miniHA™ was used in combination with HMWHA, the moisturizing effect was greater than that of either alone, indicating that the combination of miniHA and HA has a synergistic effect on the moisturizing ability of human skin. Mini-HA has a very small molecular weight (less than 10 kDa), so it can penetrate the skin and is not sticky even at high concentrations.

[0440] Effect of molecular weight of HA on moisturizing effect and TEWL Background: The outer layer of the epidermis, the stratum corneum (SC), contributes to skin barrier properties and has many protective functions, including contributing to the control of transepidermal water loss (Alexander, H. et al. “Research Techniques made simple: transepidermal water loss measurement as a research tool. J. Investigative Dermatol. (2018) 138(11):2295-2300 Elias, P. M. Skin barrier function. Curr. Allergy Asthma Rep. (2008) 8:299-305). Water movement across the SC is mainly controlled by smoothed keratinocytes surrounded by hydrophobic bilayer lipids, including ceramides, cholesterol, and free fatty acids. Skin permeability barrier function is important, and its impairment leads to downstream signals aimed at restoring barrier homeostasis. Transepidermal water loss (TEWL) is the amount of water that passively evaporates through the skin to the external environment due to the water vapor pressure gradient on both sides of the skin barrier. It is a measure of skin water barrier status that has been validated in both humans and mice by correlating TEWL with gravimetrically determined absolute water loss (ibid., Fluhr, JW. et al. Transepidermal water loss reflects permeability barrier status: validation in human and rodent in vivo and ex vivo (Id. citing Levin, J. and Maibach, H. The correlation between transepidermal water loss and percutaneous absorption: an overview. J. Control Release (2005) 103:291-99.) In addition to measuring water barrier function, in vivo TEWL measurements have been consistently correlated with transdermal absorption of topically applied compounds. (Id. citing Levin, J. and Maibach, H. The correlation between transepidermal water loss and percutaneous absorption: an overview. J. Control Release (2005) 103:291-99.)Therefore, TEWL measurements can be considered an indirect measure of skin permeability (both inside-out and outside-in), which is a function of the condition of the skin barrier. A stronger skin barrier, characterized by larger superficial keratocytes, an increased number of keratocyte layers (increased path length across the SC), and / or an improved intercellular lamellar lipid matrix, is associated with reduced TEWL (ibid., citing Damien, F. and Boncheva, M. The extent of orthorhombic lipid phases in the stratum corneum determines the barrier efficiency of human skin in vivo. J. Invest. Dermatol. (2010) 130:611-14).

[0441] TEWL is not measured directly, but is inferred from measuring the change in water vapor density (or flux) at the skin surface compared to a point away from the skin (ibid. citing Nilsson, GE. Measurement of water exchange through skin. Med. Biol. Eng. Comput. (1977) 15:208-16). When water loss across the SC is zero, the humidity in the air adjacent to the skin surface is the same as the ambient humidity. As water loss across the SC increases, the humidity next to the skin surface rises above the ambient humidity. This creates a humidity gradient on the skin surface that is proportional to the SC water loss (ibid. citing Imhof, RE et al. Closed-chamber transepidermal water loss measurement: microclimate, calibration and performance. Int. J. Cosmet. Sci. (2009) 31:97-118). Water vapour density measurements are made over a fixed area of ​​the SC within a fixed period of time and the units of TEWL are stated as grams of water per square metre per hour (g·m-2·h-1).

[0442] Materials: miniHA™ (molecular weight 8 kDa, batch 1012181), Hyaluronic Acid, molecular weight 270 kDa (HA-270 kDa, batch 1103031), Hyaluronic Acid, molecular weight 1630 kDa (HA-1630 kDa).

[0443] Vehicle: Cream. Equipment: Stratum corneum moisture meter CM825, Tewanmeter TM300. Subjects: 30 healthy volunteers aged 30–50 years. Methods: 3.0±0.1 mg / cm2 of control and test samples were applied to the left and right forearms, respectively (4×4×m2). Skin moisture of the test areas was measured before and after sample application at 1, 2, 4, 6 and 8 hours.

[0444] Figures 8A and 8B show the results of the moisture retention test (Figure 8A) and the TEWL test (Figure 8B). Figure 8A is a graph of stratum corneum moisture meter value (%) versus time for 0.1% miniHA, 0.1% HA-1630kDa, and HA-270kDa. As shown, the lower the molecular weight of HA, the better the moisture retention. The miniHA group had the highest value of skin hydration at each time point. Figure 8B is a graph of transepidermal water loss (TEWL) versus time for 0.1% miniHA, 0.1% HA-1630kDa, and 0.1% HA-270kDa. The data show that the higher the molecular weight of HA, the less skin moisture was reduced.

[0445] The moisturizing effect is better when miniHA (8kDa) and high molecular weight HA (HA-270kDA) are combined.

[0446] 9A and 9B show the results of a water retention test (FIG. 9A) and a TEWL test (FIG. 9B) when 0.1% miniHA and 0.1% HA-270kDA were combined.

[0447] The results shown in Figure 9A are bar graphs showing moisture retention capacity measured by a stratum corneum moisture meter before topical application of the sample and 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours after application. Before hydration, it was similar in both control and treated subjects. After administration of the test material, hydration improved at each time point.

[0448] Figure 9B shows a bar graph of transepidermal water loss (TEWL) versus time for 0.1% miniHA + 0.1% HA-270kDa. High molecular weight HA has good TEWL reduction ability by forming a film on the surface of the skin. MiniHA penetrates the skin and hydrates the epidermis and dermis. Together, the moisturizing effect is better than either alone.

[0449] Example 3. In vitro evaluation of miniHA permeability through in vitro reconstructed human skin Definition and Background: The term "full thickness skin" as used herein refers to skin consisting of the complete epidermis and dermis.

[0450] The term "partial thickness skin" as used herein refers to skin consisting of the entire dermis as well as only partial dermis.

[0451] "Reconstructed human epidermis" model. To produce and maintain the essential epidermal barrier, the main cell type of this tissue, keratinocytes, undergo proliferation and differentiation. During the progressive terminal maturation of keratinocytes, their cell morphology changes from a typical cuboid shape in undifferentiated proliferative cells anchored at the epidermal-dermal junction in the basal layer to a squamous epithelial morphology in dead cells in the cornified layer. Between these layers, morphological changes imply the taking of the shape of spinous cells in the spinous layer and the intracytoplasmic accumulation of dark structures called keratohyalin granules in the granular layer beneath the cornified barrier [Poumay, Y, Coquettte, A. "Modeling the human epidermis in vitro: tools for basic and applied research." Arch. Dermatol. Res. (2007) 298(8):361-9].

[0452] The typical epidermal organization into four layers reveals that within the keratinocytes a differentiation program is intended to generate the epidermal barrier. The appearance of the different layers results simply from the gradual maturation of this cell type within the epidermis. Since desquamation, i.e. peeling, of keratinized keratinocytes occurs periodically from the epidermal surface, the constant proliferation of cells in the lowest basal layer must be regulated to guarantee the homeostasis of the epidermal tissue, i.e. an equilibrium between the number of cells lost from the body surface and the number of new keratinocytes produced deep within the basal layer.

[0453] Keratinocytes can be cultured under submerged conditions as a monolayer or stratified layers, but below we will see that the cultured cells reconstitute the basis of the epidermis with a three-dimensional organization and grow further in conditions producing a cornified barrier when the surface of the culture is exposed to air [Ibid. citing Prunieras, M. et al. Methods for cultivation of keratinocytes with an air-liquid interface. J. Invest. Dermatol. (1983) 81:28s-33s].

[0454] In vitro, it is possible to obtain full differentiation by simply raising the cells to an air-liquid interface [ibid., citing Prunieras, M. et al. “Methods for cultivation of keratinocytes with an air-liquid interface.” J. Invest. Dermatol. (1983) 81:28s-33s]. The interface with air stimulates the synthesis of profilaggrin in the keratinocytes and thus the appearance of a granular phenotype when keratohyalin granules develop. These granules never appear under submerged culture conditions and seem to be the missing link that allows the final functional acquisition of keratinocytes during cell keratinization. In such culture conditions, keratinocytes located at the top of the granular layer leave their fully differentiated cytoskeleton (represented by aggregated intermediate filaments) or cell shell (represented by the cornified envelope formed after the activation of transglutaminase) in the human body to maintain a superficial barrier.

[0455] Growth at the air-liquid interface in vitro means supplying epidermal cells from the bottom of the reconstructed tissue through the basal layer. This is close to the in vivo situation, but it must be done without blood circulation. 3D reconstruction of the epidermis is adapted to a basal inert substrate such as a porous filter [ibid., citing Rosdy, M., Claus, LC. “Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface.” J. Invest. Dermatol. (1990) 95:409-14]. The filter provides a solid mechanical support to which the keratinocytes can bind via integrins, organize hemidesmosomes, and then stratify thanks to the formation of adhesive junctions and desomosomes, to form the typical epidermal layers. These layers can then be easily exposed to the air-liquid interface, if the medium is only present in the compartment below the filter. The diameter of the pores in the filter must be small enough to prevent migration of keratinocytes through the holes and eventual colonization on the other side of the filter.

[0456] After 14 days in culture, a stratified epidermis was formed in vivo, closely resembling the human epidermis. Morphologically, these cultures exhibited a well-stratified epithelium and keratinized epidermis, with significantly improved barrier function and metabolic activity. Differentiation markers such as suprabasal keratin, integrin β4, integrin α6, fibronectin, involucrin, filaggrin, trichohyalin, collagen types I, III, IV, V, and VII, laminin, heparin sulfate, and membrane-bound transglutaminase were found to be expressed similarly to those of the epidermis.

[0457] material Human reconstructed epidermis (shown in FIG. 10) is a 0.5 cm 2The epidermis is defined as the epidermis of the oocyte and is reconstituted by airlift culture on an inert polycarbonate filer (0.4 μm).

[0458] Human reconstructed full thickness skin (shown in FIG. 11) is a 0.5 cm 2 collagen matrix cultured with human keratinocytes and fibroblasts. 2 is defined as the total thickness of the culture medium and is reconstituted by airlift culture onto inert polycarbonate filters (0.4 μm).

[0459] method Summary: The aim of this study was to determine the penetration ability of sodium hyaluronate oligosaccharides (miniHA™, molecular weight <10,000 Da) into reconstructed human skin. To investigate the depth reached by the absorbed active, two parallel studies were performed: one on reconstructed human epidermis and one on reconstructed human full thickness skin (epidermis + dermis).

[0460] A schematic diagram of the experimental system is shown in Figure 12. The product applied to the surface penetrates the remodeled tissue, and a certain amount of the product is retained by the tissue structure.

[0461] The test products were applied to the surface of the tissue in 0.5% aqueous solution and the hyaluronic acid absorbed by the tissue within that time was evaluated by ELISA. The absorption kinetics was constructed by evaluating the hyaluronic acid absorbed by the epidermis and full thickness skin (epidermis + dermis) at the following experimental times, which represent the time after product application: T30 min, T1 hr, T2 hr, T4 hr, T8 hr, T24 hr. At the end of each experimental period, the treated tissues were washed, homogenized and used to determine the amount of hyaluronic acid absorbed into their structures.

[0462] protocol: The test products were diluted at 0.5% in 0.01M phosphate buffer (pH 7.4) and the resulting solution was applied to the tissue surface. Two (2) units were used for each experimental condition.

[0463] Test procedure 32μl / cm 2 A solution of 100 mg of hyaluronic acid was applied to two (2) tissue units for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours. At the end of the test period, the treated tissues were washed with phosphate buffer and homogenized to determine the amount of hyaluronic acid absorbed. Hyaluronic acid was measured by ELISA. The baseline amount of hyaluronic acid in untreated tissues was automatically subtracted during the test session.

[0464] The hyaluronic acid content, expressed in ng, was calculated for each sample collected at each experimental time. The percentage of absorbed product was calculated according to the amount of hyaluronic acid applied to the tissue. Absorption into the dermis is mathematically calculated by the difference between the total absorption into the full thickness skin and the total absorption into the epidermis. Results are expressed as mean data ± standard deviation.

[0465] The following Tables 5, 6, and 7 show data obtained from hyaluronic acid dosage in the epidermis and full thickness models treated with miniHA™. [Table 8] [Table 9] [Table 10]

[0466] Figure 13 is a graph of HA absorption rate versus time for the reconstructed epidermis, full thickness reconstructed skin, and dermis. The dermal results were mathematically calculated by subtracting the full thickness and epidermal absorption data.

[0467] The results of this permeation study on human in vitro reconstructed skin show that miniHA hyaluronan is able to penetrate the reconstructed tissue during the monitored experimental period, with a constant time-dependent absorption gradient through full thickness skin, reaching 69.5% absorption.

[0468] The results also show that hyaluronic acid is distributed through the epidermis and dermis. Absorption in the epidermis has a linear pattern until T=4 hours, while it reaches a plateau (i.e., reaches maximum absorption potential) from 4-24 hours. In the dermis, HA absorption has a linear pattern during the monitored experimental period. At the end of the monitored experimental period, HA is evenly distributed between the epidermis and dermis.

[0469] Example 4. In vitro evaluation of the wound healing potential of the three formulations after treatment of human keratinocytes summary The objective of this study was to determine the regenerative and stimulatory effects of test samples compared to human epidermal growth factor (hEGF) after 24 hours of treatment of human keratinocytes (HaCaT) using a wound healing (scratch) assay.

[0470] Test sample. Test samples were stored at room temperature. Dilutions were freshly prepared before use in cell culture. [Table 11] [Table 12] [Table 13]

[0471] method: Reagents: HaCaT cells, distilled water (Braun); DMEM medium 1g / L glucose (GIBCO); phosphate buffered saline Sigma); trypan blue solution (BioRad); DMSO (Sigma-Aldrich); Trupsin (Sigma); penicillin-streptomycin (GIBCO); L-glutamine (Sigma); EGF (Sigma-Aldrich).

[0472] FIG. 14 is a microscopic image of human hepatocytes (HaCaT line) in culture used in the study.

[0473] MTT cell viability assays were performed to select two or three working concentrations of the triplicate test samples for the following cell culture treatments.

[0474] The in vitro scratch assay was used to study cell migration in vitro. [Todaro, GJ et al. The initiation of cell division in a contact-inhibited mammalian cell line. J. Cell Physiol. (1965) 66: 325-33]. This method is based on the observation that when a new artificial gap or "scratch" is created on a confluent cell monolayer, the cells at the edge of the newly created gap migrate toward the opening to close the "scratch" until new cell-cell contacts are established. One of the main advantages of this simple method is that it mimics cell migration in vivo to some extent. For example, removing a portion of the endothelium in a blood vessel induces the migration of endothelial cells (EC) to the detached area to close the wound [Haudenschild, C. et al. Endothelial regeneration. II. Restitution of endothelial continuity. Lab. Invest. (1979) 41: 407-18]. Furthermore, the pattern of migration as loosely connected populations (e.g., fibroblasts) or as sheets of cells (e.g., epithelia and EECs) mimics the behavior of these cells during migration in vivo. The scratch assay is particularly suitable for studying the regulation of cell migration by cellular interactions with the extracellular matrix (ECM) and cell-cell interactions.

[0475] For wound healing assays, HaCaT keratinocytes were seeded overnight at 80% confluence (190,000 cells per well) in DMEM medium supplemented with 10% fetal bovine serum (FBS). The next day, a 2 mm wound was created on top of the confluent monolayer using a 1 ml pipette tip. Test samples were added to HaCaT cells and supplemented with 0.5% FBS. The untreated control was 0.5% FBS.

[0476] Images were taken before and after 24 hours of treatment and the wound area was quantified using ImageJ software. All data were statistically analyzed.

[0477] Results. Treatment with RS-0198A at 0.00001%, 0.0001% and 0.001% for 24 hours significantly enhanced wound healing by 63.4±15.7%, 61.4±17.0% and 61.9±15.7% compared to the untreated control. Treatment with RS-0198B at 0.001% for the same period induced a wound healing effect with an increase of 53.7±15.7% compared to the untreated control. RS012221 at 0.01% significantly improved wound healing in human keratinocytes by 51.7±16.9% compared to the untreated control. The positive control hEGF at 20ng / ml also promoted wound healing by 60.4±15.7% compared to the untreated control.

[0478] Conclusions. Each of RS-0198A, RS-0198b and RS12221 demonstrated regenerative and stimulatory capabilities with a significant improvement in wound healing to a similar extent as hEGF.

[0479] procedure Cell viability-MTT assay For seeded cells, cell number and viability were determined using trypan blue staining and cells were counted in a Burker chamber under a microscope. For the MTT assay, the ECVAM guidelines established by the eCVAM Database Service for alternative methods to animal testing (MTT assay protocol nr. 17) were followed.

[0480] HaCaT cells were cultured overnight in 96-well plates at a density of 10,000 cells / well in supplemented growth medium. After 24 hours, the medium was replaced with fresh medium with test samples RS-0198A, RS-0198B and RS-012221 at eight different concentrations (3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003%, and 0.001%). Untreated controls contained medium only. After 24 hours of incubation, the medium was removed and MTT solution was added to each well. The plates were incubated at 37°C for 3 hours. MTT reactivity was removed and DMSO 100% was added to each well to solubilize formazan crystals before absorbance measurements at 550 nm and 620 nm as a reference for the scanning multiwall spectrophotometer.

[0481] Eight technical replicates per condition and 16 technical replicates for untreated controls were used. All data were statistically analyzed using ordinary one-way ANOVA. Statistical significance was set at p-value <0.05 with 95% confidence. Absorbance values ​​lower than those of control cells indicated a decrease in the rate of cell proliferation. Conversely, higher absorbance values ​​indicated an increase in cell proliferation.

[0482] wound healing For seeded cells, cell number and viability were determined using Trypan-Blue staining, and cells were counted in a Burker chamber under a microscope.Human keratinocytes (HaCaT cells) were seeded overnight in DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0483] Parallel lines were drawn on the bottom of the wells (3 mm separation lines outside the plate). These lines were used as the boundaries for the scratch images, thus providing an accurate analysis in each well. Cells were seeded at 190,000 cells / well to promote the formation of a monolayer and maintained at 37°C for 24 hours. A 2 mm wide wound was then created in the confluent monolayer. The wells were washed once with PBS and refilled with DMEM+0.5% FBS. Basal images were immediately captured using a microscope. Table 11 shows the test concentrations of each test sample added to each well. Additionally, 20 ng / ml human epidermal growth factor (hEGF) was included as a positive control and 0.5% FBS as a basal control. [Table 14]

[0484] The plates were placed in a 37°C incubator and incubated for 24 hours to allow the scratches to heal....

Claims

1. 1. A cosmetic composition formulated for topical application, comprising: a. an aqueous gel component containing hyaluronic acid; b. a plant material component comprising decarboxylated cannabidiol (CBD), representing approximately 20% of the isolate; c. a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid; the composition is a slightly viscous, non-occlusive aqueous liquid; the pH of the finished product is in the range of about 4.0 to about 5.0, inclusive; and The cosmetic composition, wherein the composition is not psychoactive.

2. 2. The cosmetic composition of claim 1, wherein the hyaluronic acid comprises from about 0.10 to about 0.50 wt. % (inclusive) of low molecular weight hyaluronic acid (LMW HA) and from about 0.50 to about 1.50 wt. % (inclusive) of high molecular weight hyaluronic acid (HMW HA), wherein the ratio of the HMW HA to the LMW HA ranges from 1:0.07 to 1:1 (inclusive).

3. 2. The cosmetic composition of claim 1, wherein the molecular weight of the HMW HA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa.

4. 2. The cosmetic composition of claim 1, wherein the molecular weight of the LMW HA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 10 kDa, at least 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa.

5. 10. The cosmetic composition of claim 1, wherein the decarboxylated CBD, representing 20% ​​of the isolate, is in the form of a THC-free nano-infused water-soluble powder.

6. The cosmetic composition of claim 1, wherein the cosmetic stabilizing system comprises from about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate and from about 0.05% to about 0.50% by weight (inclusive) of arginine anisate.

7. 10. The cosmetic composition of claim 1, wherein the viscosity of the composition is in the range of 5000 to 7500 centipoise, inclusive, at room temperature.

8. 10. The cosmetic composition of claim 1, wherein the composition comprises from about 1.0% to about 5.0% by weight (inclusive) of a THC-free nano-infused water-soluble powder comprising about 20% decarboxylated CBD.

9. 1. A cosmetic composition for use in promoting and maintaining vaginal and vulvar tissue vitality and health, comprising: a. an aqueous gel component containing hyaluronic acid; b. a plant material component comprising decarboxylated cannabidiol (CBD), representing approximately 20% of the isolate; c. a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid; the composition is a slightly viscous, non-occlusive aqueous liquid; the pH of the finished product is in the range of about 4.0 to about 5.0, inclusive; and The cosmetic composition, wherein the composition is not psychoactive.

10. 10. The cosmetic composition of claim 9, wherein the parameters of vaginal / vulvar tissue vitality include one or more of improved tissue strength, proper vaginal pH, reduced susceptibility to trauma / mechanical injury, reduced inflammation, reduced itching, improved wound healing, and improved tissue elasticity.

11. wherein the composition, compared to an untreated control, a. Regulate vaginal pH, or b. Improve healing and rejuvenation of injured tissue, or c. Reduces susceptibility to trauma or mechanical injury, or d. Reduce the symptoms of trauma, injury, or disability; or e. reducing the clinical signs of dryness and inadequate hydration (e.g., loss of elasticity, inflammation); or f. Reduce itching, or The cosmetic composition of claim 10, which exhibits a combination thereof.

12. (i) Improving healing and rejuvenation of injured tissue comprises improving tissue strength. (ii) symptoms of trauma, injury, or injury include one or more of dryness, burning, irritation, discomfort, or pain; or (iii) clinical signs of dryness and inadequate hydration, including loss of elasticity, inflammation, or both; The cosmetic composition according to claim 11.

13. 10. The cosmetic composition of claim 9, wherein the hyaluronic acid comprises from about 0.10% to about 0.50% by weight (inclusive) of low molecular weight hyaluronic acid (LMW HA) and from about 0.50% to about 1.50% by weight (inclusive) of high molecular weight hyaluronic acid (HMW HA), wherein the ratio of the HMW HA to the LMW HA is in the range of 1:0.7 to 1:1 (inclusive).

14. 10. The cosmetic composition of claim 9, wherein the molecular weight of the HMW HA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa.

15. 14. The cosmetic composition of claim 13, wherein the molecular weight of the LMW HA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 10 kDa, at least 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa.

16. 10. The cosmetic composition of claim 9, wherein the decarboxylated CBD is in the form of a THC-free nano-infused water-soluble powder.

17. 10. The cosmetic composition of claim 9, wherein the cosmetic stabilizing system comprises from about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate and from about 0.05% to about 0.50% by weight (inclusive) of arginine anisate.

18. 10. The cosmetic composition of claim 9, wherein the viscosity of the composition is in the range of 5000 to 7500 centipoise, inclusive, at room temperature.

19. 10. The cosmetic composition of claim 9, wherein the composition comprises from about 1.0% to about 5.0% (inclusive) of a THC-free nano-infused water-soluble powder containing about 20% decarboxylated CBD.

20. 1. A cosmetic composition for promoting and maintaining perianal tissue vitality and perianal tissue health, comprising: a. an aqueous gel component containing hyaluronic acid; b. a plant material component comprising decarboxylated cannabidiol (CBD), representing approximately 20% of the isolate; c. a cosmetic composition stabilizing system comprising arginine, p-anisic acid, and levulinic acid; the composition is a slightly viscous, non-occlusive aqueous liquid; the pH of the composition is in the range of about 4.0 to about 5.0, inclusive; and The cosmetic composition, wherein the composition is not psychoactive.

21. wherein the composition, compared to an untreated control, a. improve healing and rejuvenation of injured tissue, or b. reducing the susceptibility of external hemorrhoidal tissue to trauma or mechanical injury, or c. Reduce the symptoms of trauma, injury, or disability; or d. reducing the clinical signs of dryness and inadequate hydration of external hemorrhoidal tissue, or e. Reduce itching, or f. The cosmetic composition of claim 20, which exhibits a combination thereof.

22. (i) improving the healing and rejuvenation of the external hemorrhoidal tissue includes improving tissue strength; (ii) symptoms of trauma, injury, or injury include one or more of dryness, burning, irritation, discomfort, or pain; or (iii) clinical signs of dryness and inadequate hydration, including loss of elasticity, inflammation, or both; The cosmetic composition of claim 21.

23. 21. The cosmetic composition of claim 20, wherein the hyaluronic acid comprises from about 0.10% to about 0.50% by weight, inclusive, of low molecular weight hyaluronic acid (LMW HA) and from about 0.50% to about 1.50% by weight, inclusive, of high molecular weight hyaluronic acid (HMW HA), and the ratio of the HMW HA to the LMW HA ranges from 1:0.7 to 1:1, inclusive.

24. 24. The cosmetic composition of claim 23, wherein the molecular weight of the HMW HA is at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 kDa.

25. 24. The cosmetic composition of claim 23, wherein the molecular weight of the LMW HA is at least 0.1 kDa to less than 10 kDa, at least 0.5 kDa to less than 10 kDa, at least 1 kDa to less than 10 kDa, at least 2 kDa to less than 10 kDa, at least 3 kDa to less than 10 kDa, at least 4 kDa to less than 10 kDa, at least 5 kDa to less than 10 kDa, at least 6 kDa to less than 10 kDa, at least 7 kDa to less than 10 kDa, at least 8 kDa to less than 10 kDa, or at least 9 kDa to less than 10 kDa.

26. 21. The cosmetic composition of claim 20, wherein the decarboxylated CBD is in the form of a THC-free nano-infused water-soluble powder.

27. 21. The cosmetic composition of claim 20, wherein the cosmetic stabilizing system comprises from about 0.25% to about 1.00% by weight (inclusive) of arginine levulinate and from about 0.05% to about 0.50% by weight (inclusive) of arginine anisate.

28. 21. The cosmetic composition of claim 20, wherein the viscosity of the composition is in the range of 5000 to 7500 centipoise, inclusive, at room temperature.

29. 21. The cosmetic composition of claim 20, wherein the composition comprises from about 1.0% to about 5.0% (inclusive) of a THC-free nano-infused water-soluble powder containing about 20% decarboxylated CBD.

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