Ophthalmic topical preparations containing helenalin, 11α,13-dihydrohelenalin, and flavonoids derived from Arnica montana extract and sodium hyaluronate, and their use in humans for the treatment of dry eye syndrome

Abstract

The present invention relates to an ophthalmic topical formulation containing Arnica montana extract and sodium hyaluronate for use in the treatment of dry eye syndrome, which improves tear film quality and regulates inflammatory processes and symptoms associated with dry eye syndrome.

Description

[Technical Field]

[0001] The present invention relates to the fields of biotechnology and medicine and consists of an ophthalmic topical formulation containing helenalin, 11-alpha, 13-dihydrohelenalin and flavonoids from Arnica Montana extract in combination with sodium hyaluronate for the primary and / or adjunctive treatment of dry eye syndrome in humans. [Background technology]

[0002] Arnica montana has been widely used in herbal medicine and homeopathy for decades. Also known as wolf bone, mountain tobacco, or leopard energy, it has been used to treat a variety of conditions, including rheumatoid arthritis, systemic lupus erythematosus, hematomas, and general inflammatory conditions. Native to Central and Southern Europe, this plant quickly spread to Asia and the Americas, where it has been used as a key ingredient in several treatments. It is an erect-stemmed plant, reaching heights of 15–60 cm, with its oval leaves forming a basal rosette at the ground. The stems are hollow, hairy, and coarse, with large, yellow flowers at the end. The branches are simple, erect, and hairless, with opposite pairs of stems, each about 30 cm tall, and terminating in a yellow flower. The leaves are oval and plantain-like, with a rough upper surface and a hairless underside.

[0003] In many Western countries (including Mexico), the word "arnica" is widely associated with the treatment of the aftereffects of trauma, and topical dermatological preparations are preferred by the general public. Currently, no reports or studies have shown adverse effects with topical use, and homeopathic and topical preparations are recommended by surgeons to aid in patient treatment to alleviate postoperative internal bleeding, edema, hematoma, and / or pain. These positive effects are related to the fact that Arnica montana contains phenolic compounds such as helenalin, 11-alpha, 13-dihydrohelenalin, and various flavonoids. However, no topical ophthalmic preparations exist, which form the basis of this invention. This medicinal plant contains flavonoids, the most important of which are quercetin, isoquercetin, rutin, campherol-3-O-glucoside, and apigenin-7-O-glucoside. It also contains other constituents such as phenolic acids, e.g., helenalin, 11-α,13-dihydrohelenalin, bitter components and amazine, which give the herb anti-inflammatory, antioxidant, analgesic, rubefacient and astringent properties.

[0004] Dry eye syndrome (OSS) is one of the most common clinical conditions worldwide, with an estimated prevalence of 9-30% of the population. However, several scientific reports have shown that the widespread use of computers and monitors, especially since the SARS-CoV-2 pandemic, has increased the impact of visual fatigue related to dry eye-related visual function in up to 70.8% and 18.3% of people who use protective masks (mouth covers), resulting in mask-associated dry eye.

[0005] Today, artificial tear drops are the preferred treatment approach (gold standard) for DES. However, adherence and compliance to treatment is only 10.2%, and 6 in 10 patients use the drops only when needed to relieve the subjective symptoms caused by DES. This leads these patients to seek home remedies in up to 76% of cases, resulting in frustration as dry eye is perceived as less than an "old person's disease" or seasonal allergies. For example, various treatment approaches are being investigated to prevent low adherence, reduce DES symptoms, and increase patient comfort.

[0006] In particular, many ocular surface diseases, including DES, are characterized by significant overproduction of reactive oxygen species (ROS), oxidative stress, and underlying inflammatory mechanisms. The hyperosmolality characteristic of DES increases the expression of proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs). Among the MMPs that are produced in excess during ocular surface hyperosmolarity are MMP-2, MMP-3, and especially MMP-9, the latter of which is the most relevant MMP in clinical testing (Quidel, Test InflamDry®). On the other hand, ROS cause oxidative damage to cells, which also contributes to inflammation. DES-associated ocular surface inflammation is usually chronic and involves activation of oxidative stress pathways, resulting in increased expression of related cytokines, such as TNF-α, IL-1β, vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and nuclear factor κB.

[0007] Along similar lines, several studies have been used to evaluate ocular surface inflammation and abnormal changes in its morphology. Ocular surface impression cytology is a noninvasive and accurate method for assessing corneal and conjunctival morphological abnormalities. For this purpose, samples are collected using a Millipore filter, and Papanicolaou staining is used to evaluate the epithelium and / or basement membrane. This method allows for the observation of the morphology of the epithelium, limbal stem cells, and conjunctival cells (goblet cells), as well as the presence and number of inflammatory cells. Importantly, the more inflammation present on the ocular surface due to DES, the greater the morphological abnormalities that will be apparent and can be analyzed by this test.

[0008] Arnica montana has significant anti-inflammatory potential. In 2011, Huber et al. demonstrated that the molecular mechanism of sesquiterpene lactones (SLs) differs from that of nonsteroidal anti-inflammatory drugs (NSAIDs), namely indomethacin and acetylsalicylic acid, because these lactones significantly reduce NF-κB-mediated inflammation. Phosphorylation and denaturation of IκB, an inhibitory subunit of NF-κB, stimulates NF-κB production. NF-κB activation by T cells, B cells, and epithelial cells is inhibited by helenalin and 11α,13-dihydroxyhelenalin, the most predominant and related SLs found in Arnica montana, which then inhibit gene expression that drives NF-κB production. This inhibition is precise and is due to alterations in the NF-κB / IκB complex via the inhibition of IκB release by helenalin and 11-α,13-dihydrohelenalin.

[0009] Arnica montana has been studied for its anti-inflammatory potential, and scientific literature has shown that it significantly reduces inflammation and edema in rats by inhibiting the action of histamine and increasing vascular permeability. Similarly, this study demonstrated that injection of Arnica montana solution, dexamethasone, or a 5% hydroalcoholic solution into these rats induced anti-inflammatory activity. In 2011, Kawakami et al. reported the presence of a series of positive inflammatory cells in rats, including CD54 (ICAM-1), CD18 (beta-2 integrin), CD45RA (B lymphocytes), CD3 (T lymphocytes), CD163 (ED2 protein), and MAC387 (monocytes and macrophages), which play important roles in the inflammatory process. It was concluded that rats with chronic edema experienced reduced edema, decreased mast cell degranulation, and increased lymphatic vessel diameter after exposure to Arnica montana. Another study concluded that acute non-fibroid mastitis disease could be effectively treated with arnica when taken orally in combination with Healwell VT-6 (containing Calcium fluoricum 200CH, Conium 30CH, Silicea 30CH, Phytolacca 200CH, Ibelladonna 30CH, Ipecacuanha 30CH, and Bryonia 30CH). Clair et al. reported that when administered with herbs such as rue, willow bank, St. John's wort, and comfrey, the use of arnica montana has therapeutic properties by increasing musculoskeletal healing in cases of deep pain such as arthritis or within the first 24–48 hours of trauma. Similarly, arnica montana extracts exhibit significant antioxidant activity. The antioxidant capacity of this plant was measured using the DPPH (2,20-diphenyl-1-picrylhydrazyl radical) free radical scavenging method and the phosphomolybdic acid method. At a concentration of 5 mg / ml, Arnica montana showed a removal rate of 71.52% in the DPPH method and a total antioxidant activity of 63.68% (phosphomolybdic acid method), which was mainly attributed to the presence of phenolic compounds such as flavonoids and quercetin.Many studies have demonstrated the anti-inflammatory activity of quercetin, which inhibits the production of tumor necrosis factor alpha (TNF-α), interleukins (IL-8, IL-1α), inflammatory enzymes (cyclooxygenase and lipoxygenase), and inhibits cytokines, interferons, and MMPs, including MMP-9. 2+ and inorganic phosphate-induced mitochondrial oxidative stress, and / or Fe 2+ The effect of Arnica montana on citrate-mediated lipid peroxidation was evaluated by measuring changes in oxygen consumption using mitochondrial suspensions prepared from the livers of male Wistar rats. The use of Arnica montana significantly reduced mitochondrial O2 consumption and increased Ca 2+ and inorganic phosphate-induced hepatic mitochondrial membrane permeability, Fe 2+ Protection was provided against citrate-mediated lipid peroxidation and ROS-mediated protein fragmentation.

[0010] Meanwhile, extraction methods for the active ingredients contained in Arnica montana vary greatly depending on the formulation and manufacturer. Many of these methods remove active ingredients such as helenalin, 11-α,13-dihydrohelenalin, and flavonoids during the Arnica montana extraction process. To maximize the yield of the active ingredients contained in Arnica montana and prioritize their anti-inflammatory properties, topical ophthalmic formulations containing Arnica montana extract should utilize ethanol extraction, which allows for concentrations of helenalin and 11-α,13-dihydrohelenalin greater than 4%. This is important because, according to the European Pharmacopoeia, all products based on Arnica montana with low concentrations of helenalin and / or 11-α,13-dihydrohelenalin lack pharmacological activity and are classified as homeopathic products, which do not provide clinical evidence of their function in the tear film in humans with dry eye syndrome. Finally, the sterilization methods used in these formulations are also crucial for preserving these active ingredients and their properties.

[0011] Sodium hyaluronate has been used as a topical ophthalmic lubricant compound in a number of commercial products for the treatment of dry eye, including Dropstar® TG (Farmigea, Italy), VISMED® (Chemedica, Switzerland), Vishibe® (Thea, USA; Chemedica, Switzerland), Hyasol (manufactured by Lab Cassara SRL for Bausch & Lomb Argentina SRL), Hyalisti (Sifi, Italy), Lalurex Ipotonico (Fidia, Italy), Hialudorf (PJbairnaDorf, Argentina), Qxyal (Santen, Germany), Lacripharma (ICN Argentina), Dunason® (Alcon Laboratorios De Brasil, Ltd.), and Maxus (Bausch & Lomb Argentina), whose functions are widely documented in the literature, but which lack anti-inflammatory and antioxidant functions.

[0012] Similarly, the use of preservative-free formulations is a favorable trend in dry eye treatment products because, in some people, dry eye can be exacerbated by the use of preservatives in topical ophthalmic formulations. For the above reasons, sterilization methods and techniques for bottles and / or containers used to store and contain the formulations are very important to achieving preservative-free formulations.

[0013] As mentioned above, DES (Dry Eye Syndrome) has a highly related inflammatory and oxidative component, which requires a wide variety of strategies to contribute to its treatment. The use of preparations with anti-inflammatory and antioxidant properties combined with lubricating action is of great interest to the scientific community.

[0014] Based on an analysis of the prior art literature, there are inventions and products that attempt to solve similar problems, such as Arnistile Eye Drops, which are commercially available in Europe. The formulations characterizing these eye drops show notable differences compared to the formulations supporting this invention, particularly the presence of 0.1% glycerin and a non-therapeutic concentration of 0.1% Arnica montana. Arnica montana extract-containing formulations (including ophthalmic formulations) commercially available in Europe are primarily based on traditional formulations with very limited clinical evidence from the European Medicines Agency, due to the lack of clinical trials supporting them. Similarly, such products lack formulations that focus on the use of helenalin, 11-α, 13-dihydrohelenalin, and flavonoids from the extract, and lack evidence of their use and effectiveness in dry eye syndrome through objective evidence such as MMP-9 measurement and / or impression cytology. Based on the above, the design and development of a formulation containing appropriate therapeutic concentrations of helenalin, 11α,13-dihydrohelenalin and flavonoids derived from Arnica montana, which supports a tolerability, safety and efficacy profile in results obtained through clinical trials with objective tests such as MMP-9 measurement and / or impression cytology in patients with active symptoms related to dry eye syndrome, remains an unsolved problem, which the formulation of the present invention solves for the first time.

[0015] Patent Document 1, entitled "PRESERVATIVE-FREE COMPOSITION FOR TOPICAL USE INCLUDING HYALURONIC ACID," was granted on March 19, 2019, and describes the topical dermatological use of hyaluronic acid as a method for preventing or treating dry eye in patients by administering it to the eyelids. It is important to clarify that this patent does not describe administration to the ocular surface, but only topical administration to the eyelids (skin). The patent further describes the possible presence of additional bioactive compounds, such as vitamin A and sucralfate. However, it does not disclose any use of bioactive Arnica montana extract. It is clear that this document provides no evidence of the efficacy of the formulation protected by the above-cited patent on inflammatory molecular processes associated with dry eye syndrome and fatigue due to monitor use.

[0016] Patent Document 2, entitled "EYE CREAM FORMULA," dated April 20, 2018, describes a moisturizing cream cosmetic for skin (eyelid) treatment that is applied to dark circles under the eyes to improve the appearance of wrinkles. The cream is used as a thin, protective layer on the skin around the eyes; however, the cream is not designed for ophthalmic use and is clearly not designed for administration to the ocular surface. The formulation in this patent contains numerous active compounds that are not used to treat dry eye syndrome and fatigue caused by monitor use. The patent does not describe the inhibition of inflammatory processes on the ocular surface that depend on the nuclear transcription factor NF-κB, or matrix metalloproteinases, which play an important role in the expression of inflammatory cytokines and inflammation and symptoms associated with dry eye syndrome.

[0017] Patent Document 3, granted on February 25, 2022, was filed by the same inventors as the present invention. The difference between the two inventions is that Patent Document 3 exclusively discloses the characteristics of a formulation, while the new application aims to improve tear film quality and regulate inflammatory processes and associated symptoms, as well as "visual fatigue" ("computer vision syndrome" or "eye strain") caused by monitor use. Therefore, the present invention can be considered an extension of Patent Document 3 in terms of its application. The present invention describes the application of the formulation described in Patent Document 3, using an ophthalmic topical formulation containing helenalin, 11α,13-dihydrohelenalin, and flavonoids derived from Arnica montan extract in combination with sodium hyaluronate for the primary and / or adjunctive treatment of dry eye syndrome in humans to improve tear film quality and regulate inflammatory processes and symptoms associated with dry eye syndrome. The active compounds in this formulation, helenalin and 11α,13-dihydrohelenalin, derived from Arnica montana extract (Arnica montana extract 100 mg / mL), selectively suppress the expression of proinflammatory cytokines (IL-1, IL-2, IL-6, IL-8, and TNF-α) and matrix metalloproteinases (MMPs) associated with the ocular surface inflammatory process responsible for dry eye syndrome (DES) symptoms and resulting from the use of monitors, by blocking the nuclear transcription factor NF-κB. A universally accepted and available method for measuring this effect is the quantification of matrix metalloproteinase-9 (MMP-9) (InflamDry MMP-9 test) from the commercial company Quidel.

[0018] Therefore, the present invention comprises a topical ophthalmic formulation containing helenalin, 11-α,13-dihydrohelenalin, and flavonoids derived from Arnica montana extract in combination with sodium hyaluronate for primary and / or adjunctive treatment of dry eye syndrome in humans. Clinical trials were conducted in subjects with mild to moderate dry eye and active symptoms to demonstrate its effects on the tear film and modulation of inflammatory processes and symptoms associated with dry eye syndrome. The technical details of the present invention are described below. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] U.S. Patent No. 10,231,990 [Patent Document 2] Chinese Patent No. 107929198 [Patent Document 3] Mexican Patent No. 390524 Summary of the Invention [Problem to be solved by the invention]

[0020] The present invention provides an ophthalmic topical formulation for use in humans that has anti-inflammatory and antioxidant properties combined with lubricating properties. The present invention comprises an ophthalmic topical formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids derived from an extract of Arnica montana, combined with sodium hyaluronate, for primary and / or adjunctive treatment of dry eye syndrome in humans. [Means for solving the problem]

[0021] The present invention is useful for improving tear film quality and modulating inflammatory processes and symptoms associated with dry eye syndrome, including: i. A lubricant containing ingredients having anti-inflammatory and antioxidant properties, and helenalin, 11-alpha, 13-dihydrohelenalin and flavonoids from Arnica montana extract, and sodium hyaluronate, wherein each mL contains: ii. Arnica montana extract: 100~500mg; iii. Sodium hyaluronate: 1-4 mg. [Brief explanation of the drawings]

[0022] [Figure 1]The design of a tolerability and safety study is presented, in which the test formulation is a topical ophthalmic formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids from Arnica montana extract in combination with sodium hyaluronate. [Figure 2] The efficacy study design is shown. During the study, all subjects participated in a baseline visit and were assigned to one of two groups. In addition, all subjects had a minimum follow-up period of 4 weeks (28 days). [Figure 3]

[0023] Figures 1A-1C show images from a safety study. The images show the same eye of the same patient at baseline (A) with (blue light) and without (white light) shots, week 1 (B), and week 3 (C). These images show that there was no change in ocular surface staining after use of the test formulation. [Figure 4] A flowchart of the efficacy study is shown; the flowchart shows subjects who were enrolled, accepted, randomized, and analyzed in both groups (Group A = test formulation and Group C = ocular lubricant (Eyestil lub® control) in the efficacy study. [Figure 5] FIG. 1 is a schematic diagram of the impression cytology procedure used by the inventors. [Figure 6] Impression cytology images at baseline (1-A) and one month (1-B) in the study group are shown. All patients showed statistically significant improvement. [Figure 7] Impression cytology images at baseline (2-A) and one month (2-B) in the control group are shown. None of the patients showed improvement, and some even worsened. DETAILED DESCRIPTION OF THE INVENTION

[0023] This paper describes a topical ophthalmic formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids derived from Arnica montana extract, combined with sodium hyaluronate, for the primary and / or adjunctive treatment of dry eye syndrome in humans. Additional objectives include improving tear film quality and regulating inflammatory processes and symptoms associated with dry eye syndrome. These positive effects are clearly demonstrated in the following description and the accompanying illustrative figures, which serve as references for the studies conducted.

[0024] It is an object of the present invention to provide a topical ophthalmic formulation for use in humans that has utility for improving tear film quality and modulating inflammatory processes and symptoms associated with dry eye syndrome.

[0025] The formulation comprises: a) A lubricant containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids from Arnica montana extract in combination with ingredients having anti-inflammatory and antioxidant properties and sodium hyaluronate for the primary treatment and / or as an adjunct for the treatment of dry eye syndrome in humans, wherein each mL contains: i. Arnica montana extract: 100~500mg; ii. Sodium hyaluronate: 1-4 mg.

[0026] Furthermore, it is important to mention that the present formulation is protected by a patent by the inventors of this novel invention, the object of which is its use and application for improving tear film quality and modulating inflammatory processes and symptoms associated with dry eye syndrome.

[0027] The applications and uses of the formulation include, and a series of tests have been carried out to demonstrate, the following applications and uses: · Anti-inflammatory, antioxidant and lubricating properties that relieve symptoms associated with dry eye syndrome and monitor fatigue (computer vision syndrome or eye strain); Furthermore, it has the potential to improve vascular permeability through modulation of histamine and to modify the inflammatory cascade through selective inhibition of transcription factors; Furthermore, it has the potential to improve vascular permeability through modulation of histamine and to modify the inflammatory cascade through selective inhibition of transcription factors.

[0028] A clinical trial to test the use of a lubricant containing helenalin, 11α,13-dihydrohelenalin, and flavonoids from Arnica montana extract in combination with sodium hyaluronate in the treatment of dry eye syndrome and fatigue due to monitor use - computer vision syndrome - (computer vision syndrome or eye strain).

[0029] To validate this application and use of the formulation, favorable, unexpected, and previously unreported results of a clinical trial in humans of a topical ophthalmic formulation with lubricating properties as well as anti-inflammatory and antioxidant properties are presented. The present invention comprises a topical ophthalmic formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids derived from Arnica montana extract in combination with sodium hyaluronate for use in the treatment of dry eye syndrome. The present invention is useful for improving tear film quality and regulating inflammatory processes and symptoms associated with dry eye syndrome, including:

[0030] A formulation for use in improving tear film quality and modulating inflammatory processes and symptoms associated with dry eye syndrome in humans comprises, on a 1 mL basis: a) Arnica montana extract: 100-500mg, b) Sodium hyaluronate: 1-4 mg. Including, The formulation has anti-inflammatory, antioxidant and lubricating properties.

[0031] To confirm these positive effects on the ocular surface, a prospective, randomized, double-blind Phase I-II clinical trial was conducted using two test groups and a control group (placebo). Furthermore, to confirm the range of Arnica montana extract, dilutions of the initial concentration were performed in 1 ml doses, ranging from 100 to 500 mg. The effects of Arnica montana extract were confirmed in the range of 2 to 100 mg per 1 ml of formulation.

[0032] The study was conducted in two parts. The first part (tolerance and safety study) was conducted in a single, prospective, blinded study group of 20 healthy subjects to evaluate the tolerability and efficacy of the formulation. The second part (efficacy) of the clinical trial was conducted in 48 eyes of 48 patients diagnosed with mild to moderate dry eye syndrome who used a monitor for more than 8 hours per day and who met all inclusion and exclusion criteria of the study (OSDI > 13, tear burst time < 10 seconds, and MMP-9 positivity). The clinical trial was conducted in 2021 at a private ISO 9001:20015-accredited ophthalmic research facility in a single center in patients diagnosed with mild to moderate dry eye syndrome who used a monitor for more than 8 hours per day. Patients were divided into two distinct groups: A group using the preparation of the present invention as a first-line treatment (test group = A). A group using an ocular lubricant (commercially available 0.4% sodium hyaluronate (Eyestil lub®, Laboratorios SI FI, Mexico) as a first-line treatment (control group = C).

[0033] Prior to patient enrollment, approval from the internal review board (ethics research committee and research committee) was obtained. The study adhered to the principles of the Declaration of Helsinki.

[0034] The protocol complied with the International Conference on Harmonization of Clinical Trials guidelines and all other applicable local regulatory requirements and laws prior to enrollment, and written informed consent was obtained from all subjects after a detailed explanation of the nature of the study and possible adverse events (AEs).

[0035] Subjects over 18 years of age were diagnosed with mild to moderate dry eye syndrome by OSDI (Ocular Surface Disease Index), a non-invasive tear film break-up time (NIF-BUT) of less than 10 seconds, and any degree of corneal staining with sodium fluorescein and conjunctival staining with Lissamine Green, a positive MMP-9 measurement using the Quidel InflammaDry® kit, and were subject to visual stress and screen work >8 hours per day.

[0036] intervention: 1. Tolerance and Safety Study. Twenty subjects were included in a single study group (a test formulation based on Arnica montana extract and sodium hyaluronate), and patients were instructed to administer one drop three times daily. The intervention period was 3 weeks (21 days), and a tolerability (ocular tolerance) questionnaire was completed in all patients. The design of the tolerability and safety study is shown in Figure 1; in this case, the AcuSense drops® product was an identical topical ophthalmic formulation containing helenalin, 11-α,13-dihydrohelenalin, and flavonoids derived from Arnica montana extract combined with sodium hyaluronate.

[0037] 2. Efficacy Study. Subjects were randomly assigned to one of two study groups, including group (A) using the test formulation (formulation described herein) as the first-line treatment, or group (C) using an ocular lubricant (Eyestil lub®, Laboratorios SI FI, Mexico). Patients in both groups were instructed to apply one drop of the corresponding formulation three times a day for four weeks (28 days). During each visit, subjects underwent different tests: Symptom assessment using the Ocular Surface Disease Index (OSDI) questionnaire. This questionnaire is the most widely used by ophthalmologists to assess dry eye symptoms. A score of >13 is considered a positive criterion for the diagnosis of dry eye; Measurement of best corrected visual acuity (AVMC). Measurement of best corrected visual acuity was performed based on the ETDRS protocol, which is the most widely used visual acuity measurement protocol in clinical studies; A complete clinical eye examination using a slit lamp. This is a non-invasive test and is the most widely used by ophthalmologists to assess the overall condition of the eye. · Objective measurement of non-invasive tear break-up time (NIF-BUT) and mean time (NIAvg-BUT) using the Schwind Sirius® topography device (CSO SRL, Italy); MMP-9 sampling using the Quidel device (InflammaDry®). This is the most specific test for objectively measuring ocular surface inflammation. This test measures MMP-9, which is specific to ocular surface inflammation and is recognized as a determinant in the chronic inflammation that occurs in dry eye. If dry eye treatment is effective, this test will be negative in subsequent tests; Impression cytology sampling. This test allows the pathologist to evaluate the presence of inflammatory cells on the ocular surface (conjunctiva). If dry eye treatment is effective, the presence of these cells in the conjunctiva will be negative in subsequent measurements; Ocular surface staining with sodium fluorescein (AK-Fluor® Akorn, Lake Forest, IL, USA) and Lissamine Green (Rose Stone Enterprises, Alta Loma, CA, USA) to assess the corneal and conjunctival surfaces of the Sjögren Clinical Collaboration Alliance (SICCA). The OCT (Ocular Staining Score) scale was used. These stains are used by the ophthalmology community to assess the severity and response to treatment of dry eye syndrome; Schirmer test (Eagle Vision, Inc., Memphis TN, USA). This test is used by the ophthalmology community to assess the severity and response to treatment of dry eye syndrome; Intraocular pressure measurement (mmHg) (Icare® TA01i tonometer, Belleville, MI, USA). This test is part of the standard examination that all ophthalmologists perform on their patients; Fundus evaluation using articular fundus examination (Killer Vantage Plus LED, Malvern, PA, USA). This examination is part of the standard examination that all ophthalmologists perform on their patients.

[0038] To ensure the objectivity of these measurements, all measurements were performed by the same investigator at the same time in the morning (9:00 am to 11:00 am), in the same room, in a quiet environment, with controlled humidity (40-50%) and the same controlled temperature (23-24°C), without air conditioning during the study period. Patient compliance with the use of the study medication (including placebo) was assessed based on written diaries.

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[0039] result. 1. Tolerability and Safety Study. No adverse events were recorded throughout the entire follow-up period. The formulation was well tolerated by all study subjects (ocular tolerability questionnaire results). This study demonstrated that the formulation is tolerable and safe for use in humans. Figure 3 shows representative images from the safety study; the images show the same eye of the same patient at baseline (A) with (blue light) and without (white light) shots, week 1 (B) and week 3 (C) shots, and the results show that there was no change in ocular surface staining after use of the study formulation.

[0040] 2. Efficacy Study. The study included 48 eyes of 48 patients with mild to moderate dry eye disease (DED) who met all inclusion and exclusion criteria. Data from 48 subjects (24 for Group A and 24 for Group C) were analyzed. Therefore, all subjects completed the minimum follow-up period, and the overall subject compliance rate with treatment use was 100% (>90%). For this efficacy study, both treatment groups (Groups A and C) showed statistically significant differences between baseline and Month 1 (Day 28) in OSDI (Ocular Surface Disease Index) test results and noninvasive tear break-up time (NIF-BUT). However, only the test group (Group A) showed statistically significant differences between baseline and Month 1 (Day 28) in noninvasive mean tear break-up time (NIAvg-BUT) scores and sigma tests between baseline intake and Day 28 of follow-up. Regarding objective measurements of ocular inflammation (measurement of MMP-9 by the Quidel InflammaDry® test), the test group showed a statistically significant reduction in the number of eyes with positive tests (100% at baseline intake vs. 33.4% at follow-up day 28), whereas this did not occur in the group using an ocular lubricant (Eyestil lub®) (100% at baseline intake vs. 83% at follow-up day 28). This data is extremely important because it demonstrates the anti-inflammatory activity of the topical ophthalmic formulation of the present invention, containing helenalin, 11-α,13-dihydrohelenalin, and flavonoids derived from Arnica montana extract in combination with sodium hyaluronate, through objective and reliable measurements of ocular surface inflammation. Similarly, conjunctival impression cytology tests showed significant changes in the test group (Group A) after corresponding pathology analysis, but not in the group using an ocular lubricant (Group C). These latter two results confirm that the addition of helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids from Arnica montana extract to sodium hyaluronate confers anti-inflammatory activity that sodium hyaluronate itself does not possess (statistically significant result for Group A vs. non-significant result for Group C).

[0041] Finally, ocular surface staining studies showed a statistically significant difference in the test formulation group (Group A) but not the control group (Group C). Figure 4 graphically depicts the efficacy study, and the flow chart shows subjects in both groups (Group A = test formulation and Group C = ocular lubricant (Eyestil lub®)) who were enrolled, accepted, randomized, and analyzed in the efficacy study.

[0042] Table 1 shows the demographic and baseline data (with standard deviations) for the efficacy study. All patients in both groups demonstrated diagnostic criteria for mild to moderate dry eye syndrome, and all had a positive MMP-9 test. [Table 1]

[0043] Table 2 shows the data from the efficacy study. Subjects enrolled in the efficacy study were accepted, randomized, and analyzed in both groups (Group A = test formulation and Group C = ocular lubricant (Eyestil lub®)). Improvements in OSDI and NIF-BUT were statistically significant in both study groups (both groups used lubricant (sodium hyaluronate)).

[0044] However, only subjects in Group A showed statistically significant improvements in the NIAvg-BUT, Schirmer test, and especially the MMP-9 measurement, this latter test being specific for measuring inflammation at the ocular surface, indicating the unique and unexpected anti-inflammatory activity of the formulation.

[0045] Baseline = baseline intake, Month 1 = month 1 of follow-up, * "Statistically significant difference." [Table 2]

[0046] Table 3 shows the quantitative analysis of ocular surface staining. With regard to ocular surface staining, Group A showed a statistically significant difference, which is particularly relevant since this marker is specific to chronic damage to the ocular surface due to dry eye. Group C did not show a statistically significant improvement in corneal staining. Baseline = baseline intake, Month 1 = follow-up month 1. * "Statistically significant difference." [Table 3]

[0047] Table 4 shows the quantitative analysis of the results of the ocular surface impression cytology test, showing that the test group demonstrated a statistically significant improvement between the baseline and 28-day measurements. Similarly, the test group demonstrated a statistically significant improvement over the results obtained in the control group. Finally, the control group demonstrated no improvement between the baseline and 28-day intakes. [Table 4]

[0048] Therefore, the data of the present invention show that this topical ophthalmic formulation containing helenalin, 11-alpha,13-dihydrohelenalin, and flavonoids from Arnica montana extract in combination with sodium hyaluronate has anti-inflammatory activity and properties (statistically significant results in the Quidel InflammaDry® MMP-9 measurement test and conjunctival impression cytology test), improves tear film quality, with statistically significant results in the non-invasive tear break-up time test (NIF-BUT), mean tear break-up time (NIAvg-BUT) and Schirmer test), improves the condition of the ocular surface tissue (Lissamine Green conjunctival and sodium fluorescein corneal staining tests), reduces tear evaporation (statistically significant results in the non-invasive tear break-up time test (NIF-BUT) and mean tear break-up time (NIAvg-BUT)), and improves symptoms associated with dry eye syndrome and monitor use (statistically significant results in the OSDI test).

[0049] In summary, the topical ophthalmic formulation of the present invention, containing helenalin, 11-α,13-dihydrohelenalin, and flavonoids derived from Arnica montana extract in combination with sodium hyaluronate for the treatment of dry eye syndrome in humans, is tolerable, safe, and effective in alleviating symptoms associated with dry eye syndrome. It exhibits anti-inflammatory effects that suppress ocular surface inflammation and improve tear film quality in humans with dry eye syndrome and fatigue from monitor use (computer vision syndrome or asthenopia). Furthermore, the results confirmed the formulation's efficacy at both low and high doses of 100-500 mg of Arnica montana extract per mL. At the upper end of the dose range used, 500 mg per mL, the 500-1000 mg per mL formulation, did not produce comparable responses to those obtained with the initial formulation. For the lower limit of 100 mg per mL in the original formulation, the lower limit was used in the range of 2 to 100 mg per mL of formulation, and a response comparable to that of the initial formulation was observed. Thus, evidence is provided that Arnica montana extract can be used in the range of 2 to 100 mg per mL of formulation without affecting its properties for use in dry eye syndrome. Thus, the behavioral distribution of values ​​for Arnica montana extract can be seen to be biased toward the lower end of the range, with values ​​ranging from 2 to 500 mg per mL of formulation containing that ingredient.

[0050] Conclusion. Based on the results obtained in a clinical trial designed to evaluate tolerability, safety, and efficacy, it has become possible to demonstrate the proof of concept of topical administration of an ophthalmic topical formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids derived from Arnica montana extract in combination with sodium hyaluronate for the treatment of dry eye syndrome in humans, as well as its effect on improving tear film quality (measurements of NIF-BUT, NIAvg-BUT, Schirmer test), modulating inflammatory processes (measurements of MMP-9, corneal and conjunctival ocular surface staining, and meibomian gland inflammation), and improving symptoms associated with dry eye syndrome (OSDI).

[0051] These favorable, unexpected, and previously unreported results from clinical trials demonstrate the anti-inflammatory and antioxidant properties, as well as the improvement of tear film quality, modulation of inflammatory processes, and amelioration of symptoms associated with dry eye syndrome of the presently described formulations, which formulations include: a) A lubricant containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids from Arnica montana extract in combination with ingredients having anti-inflammatory and antioxidant properties and sodium hyaluronate, wherein each mL contains: i. Arnica montana extract: 100~500mg; ii. Sodium hyaluronate: 1-4 mg.

[0052] The formulation has an adequate safety profile, is well tolerated, has a clinically significant efficacy profile, and has anti-inflammatory effects that improve symptoms and associated signs of ocular surface inflammation, such as corneal and conjunctival de-epithelialization, tear break-up time, and meibomian gland inflammation (measured by a Schwind Sirius® device), and reduces the presence of MMP-9 on the ocular surface.

[0053] Ocular surface impression cytology: Morphologic changes in the conjunctiva. Background: Impression cytology (IC) is a technique that allows the recovery of the outermost layer of cells from the ocular surface by using various types of filters. It is a minimally invasive method to evaluate the morphology of human conjunctival epithelial cells in the diagnosis of dry eye disease, a common and painful condition associated with aging, contact lens use, autoimmune diseases, and refractive surgery (LASIK). Recently, it has been used for dry eye disease.

[0054] IC has traditionally been used to identify morphological changes (e.g., squamous metaplasia) in ocular surface diseases, primarily dry eye disease. However, subsequent IC studies have evaluated other ocular surface diseases and observed substantial loss of conjunctival goblet cells and changes in epithelial cell size. Several groups have used IC to evaluate the effects of dry eye disease treatments on the ocular surface, including the effects of preservatives and autologous serum in ophthalmic medications.

[0055] method. All subjects (test and control groups) underwent IC in the left eye. The IC procedure included membrane impression cytology.

[0056] Millipore, Papanicolaou staining, and Nelson grading. The technique used is illustrated in Figure 5. The figure shows a filter paper disc cut in half (left) and the resulting D-shaped segment (cut into orientations a and b) applied to the bulbar conjunctiva and cornea using the Goldmann tonometer head, applying uniform pressure to the filter paper (right). The 11-α,13-dihydrohelenalin content ranged from 0.004% to 5% per ml for use in dry eye syndrome.

[0057] result. Eight of the 24 subjects (33.3%) in the test group and seven of the 24 subjects (29.1%) in the control group showed abnormalities in ocular surface morphology. All subjects in the test group showed normal impression cytology one month after treatment with the test eye drops. However, none of the eyes in the control group showed improvement in impression cytology one month after treatment with the ocular lubricant.

[0058] A statistically significant improvement in ocular surface morphology was observed in the test group by impression cytology one month after treatment with AcuSense drops®. No such improvement was observed in the control group.

[0059] Table 5 shows the ocular surface impression cytology. Test group vs. control group. After one month of treatment, all eyes with ocular surface morphological abnormalities showed improvement in the test group. None of the control group eyes showed improvement. [Table 5]

[0060] Table 6 shows the statistical analysis of the study. There were statistically significant differences in impression cytology between the test group (baseline vs. 1 month) and the test group vs. the control group. The control group showed no statistically significant differences. [Table 6]

[0061] After one month of treatment with AcuSense drops®, there was an improvement in ocular surface morphology. Patients with morphological abnormalities of the conjunctival mucus and epithelium showed normal impression cytology at the end of follow-up.

[0062] Test group: Eight of 24 (33.3%) eyes in the test group showed morphological abnormalities of the ocular surface at the start of the study. All of these showed normal impression cytology after one month of treatment with AcuSense drops®. Figure 6 shows the Millipore membrane impression cytology, Nelson grading, and Papanicolaou staining.

[0063] 1A: Baseline, stage 0, grade 3. Abundant cell count, 80–200 cells / mm 2 Goblet cells, moderately frequent intercellular spaces, cytoplasmic-nuclear ratio shifted to predominantly cytoplasmic, mild inflammation, slightly increased apoptosis.

[0064] 1-B: 1 month, stage 0, grade 0. Abundant cell count, more than 400 cells / mm 2 Goblet cells are cohesive and have a normal cytoplasmic-to-nuclear ratio. No inflammatory cells or obvious apoptosis are seen.

[0065] None of the eyes with ocular surface morphological abnormalities in the control group showed improvement after 1 month of ocular lubricant treatment.

[0066] Control group: Seven of 24 (29.1%) eyes in the test group showed morphological abnormalities of the ocular surface at the start of the study. None of them showed improvement in impression cytology after one month of treatment with the ocular lubricant. Figure 7 shows the Millipore membrane impression cytology, Nelson grading, and Papanicolaou staining.

[0067] 2-A: Baseline, stage 0, grade 1. Abundant cell count, 300-400 cells / mm 2 Goblet cells, cohesive or discrete cell spacing, normal cytoplasm-to-nuclear ratio. Minimal inflammation, minimal apoptosis.

[0068] 2-B: 1 month, stage 0, grade 2. Abundant cell count, 200-300 / mm 2 Goblet cells, frequent mild cell separation, cytoplasmic-nuclear ratio shifted to a cytoplasmic predominance, mild inflammation, slightly increased apoptosis.

[0069] Conclusion. The use of the test eye drops (Arnica montana extract + hyaluronic acid) successfully improved IC. Conventional ocular lubricants did not show any improvement in morphological abnormalities in IC. These results should be considered as objective evidence of the therapeutic effect of the biological activity present in Arnica montana extract (helenalin and 11-α,13-dihydrohelenalin) on the ocular surface of subjects with dry eye disease.

[0070] Use of a validated formulation based on helenalin, 11-alpha, 13-dihydrohelenalin and flavonoids from Arnica montana extract in combination with sodium hyaluronate to improve tear film quality and regulate inflammatory processes and symptoms associated with dry eye syndrome.

[0071] With the validation presented in the previous studies, the formulation claimed in the present invention is as follows:

[0072] An ophthalmic formulation for use in treating symptoms associated with dry eye syndrome comprises, on a 1 mL basis: a) Arnica montana extract: 100-500mg, b) Sodium hyaluronate: 1-4 mg.

[0073] Additionally, this formulation has been validated for use as follows: Optimizing the presence of bioactive components present in Arnica montana through an extraction process using ethanol, ethyl ether, acetone, chloroform, and petroleum ether for use in dry eye syndrome; For use in dry eye syndrome, the content of 11-α,13-dihydrohelenalin ranges from 0.004% to 5% based on 1 mL; Blocking the nuclear transcription factor NFκB and suppressing the expression of inflammatory interleukins and matrix metalloproteinases on the ocular surface of humans with dry eye; Reduction of morphological abnormalities of the ocular surface associated with inflammatory processes present in human dry eye syndrome; Reduced tear film evaporation in people with dry eye syndrome; Reduced conjunctival de-epithelialization in humans with dry eye syndrome; Increased tear film stability in people with dry eye syndrome; Modulation of inflammatory processes associated with dry eye syndrome in humans; · Reduction of symptoms caused by inflammatory processes associated with dry eye syndrome in humans; For use in dry eye syndrome, the formulation can be sterilized by hot steam method, ultrafiltration, and gamma radiation; For use in dry eye syndrome, the formulation may contain preservatives such as benzalkonium chloride, benzyl alcohol, or may be preservative-free; For use in dry eye syndrome, the preparation may be produced as a botanical medicine; For use in dry eye syndrome, the preparation may be manufactured as a medical device; For use in dry eye syndrome, the formulation may be contained in a bottle specially designed for preservative-free topical ophthalmic products.

[0074] Furthermore, the formulation has anti-inflammatory, antioxidant, and lubricating properties in ophthalmic applications and in the treatment of dry eye syndrome. The above explanation of the definitions of the present disclosure is provided to enable those skilled in the art to implement or use the present invention. Various modifications to these definitions and / or implementations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but should be accorded the widest scope consistent with the following claims and the principles and novel features described herein.

[0075] The inventions have been fully described and are believed to be novel and, therefore, what is contained in the following paragraphs is considered to be exclusive property.

Claims

1. 1. A topical ophthalmic formulation containing helenalin, 11-alpha, 13-dihydrohelenalin, and flavonoids from an extract of Arnica montana in combination with sodium hyaluronate for the treatment of dry eye syndrome in humans, the formulation containing the following per mL: a. Arnica montana extract: 100-500 mg; b. Sodium hyaluronate: 1-4 mg. An ophthalmic topical formulation comprising:

2. 2. The topical ophthalmic preparation according to claim 1, characterized in that the biological activity of the Arnica montana extract is obtained by an extraction process using ethanol, ethyl ether, acetone, chloroform and petroleum ether for use in dry eye syndrome.

3. 2. The ophthalmic topical formulation according to claim 1, wherein the lower limit range of the Arnica montana extract is in the range of 2 to 100 mg per mL for use in treating dry eye syndrome.

4. 2. The topical ophthalmic preparation according to claim 1, wherein the content of 11-α,13-dihydrohelenalin is in a concentration ranging from 0.004% to 5% based on 1 mL for use in treating dry eye syndrome.

5. 2. The ophthalmic topical preparation according to claim 1, characterized by having anti-inflammatory, anti-oxidative and lubricating properties.

6. 2. The topical ophthalmic formulation according to claim 1, for use in blocking the nuclear transcription factor NFκB and suppressing the expression of inflammatory interleukins and matrix metalloproteinases on the ocular surface of a person with dry eye.

7. 10. The topical ophthalmic formulation of claim 1 for use in reducing morphological abnormalities of the ocular surface associated with inflammatory processes present in human dry eye syndrome.

8. 10. The topical ophthalmic formulation of claim 1 for use in reducing tear film evaporation in a person with dry eye syndrome.

9. 10. The topical ophthalmic formulation of claim 1 for use in reducing conjunctival de-epithelialization in a human with dry eye syndrome.

10. 10. The topical ophthalmic formulation of claim 1 for use in increasing tear film stability in humans with dry eye syndrome.

11. 10. The topical ophthalmic formulation of claim 1 for use in modulating inflammatory processes associated with dry eye syndrome in humans.

12. 10. The topical ophthalmic formulation of claim 1 for use in reducing symptoms caused by inflammatory processes associated with dry eye syndrome in humans.

13. 10. The topical ophthalmic preparation according to claim 1, which can be sterilized by hot steam method, ultrafiltration and gamma radiation for use in dry eye syndrome.

14. 10. The topical ophthalmic formulation of claim 1, which may or may not contain a preservative such as benzalkonium chloride, benzyl alcohol, etc., for use in dry eye syndrome.

15. 10. The topical ophthalmic formulation of claim 1, which can be contained in a bottle specially designed for preservative-free topical ophthalmic products for use in dry eye syndrome.

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