Novel eyelid delivery of ophthalmic composition containing muscarinic receptor modulators for prevention and treatment of ocular disorders

Abstract

The present invention provides a method for ocular drug delivery, comprising a step of topically applying a therapeutically effective amount of a pharmaceutical composition to an eyelid skin of a subject, wherein the pharmaceutical composition comprises: (a) a therapeutically active agent comprising a muscarinic acetylcholine receptor (mAChRs) modulator; and (b) a pharmaceutically acceptable carrier.

Description

[0001] Novel Eyelid Delivery of Ophthalmic Composition Containing Muscarinic Receptor Modulators for Prevention and Treatment of Ocular Disorders Cross-Reference to Related Applications

[0001] This application claims priority to US Application No.63 / 695,198, filed on September 16, 2024, and US Application No.63 / 799,249, filed on May 2, 2025, the contents of both of which are incorporated herein by reference in their entirety. Background of the Invention

[0002] Ocular disorders, affecting the health and function of the eyes, include a spectrum of conditions ranging from age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion to refractive errors like myopia (nearsightedness) and hyperopia (farsightedness). Other disorders include astigmatism and chronic afflictions, such as dry eye syndrome, characterized by inadequate lubrication and discomfort. Particularly, those disorders related to the posterior segment of the eye remain unmet treatment areas.

[0003] In the eye and its adnexa, muscarinic acetylcholine receptors (mAChRs) are expressed in both anterior and posterior segments (especially retina and sclera), playing a crucial role in various physiological functions. See, e.g., Caulfield, M. P., & Birdsall, N. J. (1998). International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacological reviews, 50(2), 279-290. Dysregulation or hyperactivity of muscarinic receptors in the eye can lead to various ocular disorders, such as glaucoma, myopia, accommodative dysfunctions, etc. Muscarinic receptor modulators, such as agonists and antagonists, play a significant role in treating various ocular disorders, each with distinct mechanisms of action.

[0004] For instance, atropine is a mainstay in ophthalmology for preventing myopia progression. See, e.g., A. Chierigo et al., Pharmaceutics, 2022, 14(5), 900. See, e.g., W.Y. Wang et al., Biomedicine & Pharmacotherapy, (2021) 133, 111092. Acting as muscarinic agonists, pilocarpines can reduce intraocular pressure in glaucoma through their action on muscarinic receptors. See F. Mitchelson, Muscarinic Receptors, 2012, 263-298. Scopolamine, with its muscarinic antagonist properties, is beneficial in inducing drying effects for specific ocular conditions. See, e.g., P. Yu et al., Annals of Translational Medicine, 2021, 9(4); and M.M. Rahman Int. J. Molecular Sciences, 2021, 22(22), 12102.

[0005] The therapeutic use of muscarinic receptor agonists and antagonists highlights their critical role in managing ocular disorders. However, their clinical application requires careful consideration of potential side effects and the means of administration. The development of more selective ligands for specific receptor subtypes holds promise for more precise treatments with reduced adverse effects.

[0006] Myopia, characterized by axial elongation of the eye, is a prevalent condition affecting a significant portion of the population. Traditional treatments include corrective lenses and pharmacological interventions. Muscarinic acetylcholine receptor modulators (e.g., atropine and pirenzepine) have been widely studied in both animal studies and human clinical trials. The exact mechanism underlying atropine’s efficacy in slowing eye growth is unclear. However, the synthesis of glycosaminoglycan, of the scleral extracellular matrix, was inhibited by atropine in studies involving scleral tissue from chicks. Muscarinic receptors have also been found in the retinal pigment epithelium, a layer that is involved in transferring the signaling cascade toward the target tissue - i.e., the choroid and / or the sclera. See, e.g., Upadhyay et al., Eye & contact lens, 46(3), 129–135. To elicit therapeutic effects, atropine or pirenzepine needs to reach the back of eye, with an optimal drug concentration.

[0007] Atropine is a nonselective muscarinic antagonist that has an equal affinity for all five acetylcholine receptor subtypes found in the retina. While this allows atropine to be effective in more diluted concentrations, side effects remain pervasive given its nonselective nature, affecting many parts of the eye. There have also been concerns regarding the potential long-term systemic side-effects of atropine usage. Several anticholinergic drugs and other medications with anticholinergic properties have been associated with central side-effects adversely affecting cognitive function. See, e.g., C. Fox et al., Age and ageing, 43(5), 604–615. The development of pirenzepine ophthalmic solution was an attempt to slow down the growth of the myopic eye using an alternative nightly eye drop. Pirenzepine is a more selective muscarinic receptor 1 (MR1) antagonist, which attenuates axial eye growth in a chick model of myopia compared to M2 and M3 antagonists which did not prevent axial elongation. See, e.g., E.M. Leech et al., Ophthalmic and Physiological Optics, 1995, 15(5), 351-356. This effect of pirenzepine on myopia was further extended to include the tree shrew and rhesus monkey. See, e.g., C.L. Cottriall et al., Investigative Ophthalmology & Visual Science, 1996, 37(7), 1368-1379; and M Tigges et al., Optom Vis Sci. 1999 Jun ;76(6):397-407. The relative selectivity of pirenzepine as an M1-antagonist suggested that this pharmacologic agent might prevent myopia by the same mechanism as atropine, but without the disabling side effects of mydriasis in outdoor light and loss of accommodation, which are mediated predominantly through the M3-receptor.

[0008] In a 2003 study, pirenzepine was shown to be well tolerated in children. See Bartlett, J. D., Niemann, K., Houde, B., Allred, T., Edmondson, M. J., & Crockett, R. S. (2003). A tolerability study of pirenzepine ophthalmic gel in myopic children J Ocul Pharmacol Ther. 2003 Jun;19(3):271-9. A 2008 report suggested that pirenzepine was effective for slowing refractive error progression, but did not have a statistically significant effect on axial length. See R.M. Siatkowski et al., J. Amer. Ass’n for Pediatric Ophthalmology and Strabismus, 2008, 12(4), 332- 339.

[0009] For several decades, eye drops have remained one of the most extensively utilized pharmaceutical formulations for ocular diseases. See, e.g., Przemysław Baranowski et al., Scientific World Journal.2014 Mar 18:2014:861904. Currently, eye drops account for about 90% of ophthalmic medicines, primarily due to their ease of administration and good patient compliance. However, several drawbacks are associated with the utilization of eye drops. Eye medications cannot be administered beyond the capacity of the conjunctival sac due to its limited volume. Most eye drops exhibit low bioavailability, poor targeting efficacy, and are virtually impossible to administer during sleep. Anatomical and physiological constrains such as tear turnover, nasolacrimal drainage, reflex blinking, and ocular static and dynamic barriers impede the bioavailability and controlled delivery of drugs administered as eye drops. See Ripal Gaudana, AAPS J.2010 Sep;12(3):348-6.

[0010] To overcome all the drawbacks discussed above, it is desired to have an improved approach to deliver ophthalmic drugs with high targeting ability, while simultaneously improving drug absorption into the ocular tissues. It is particularly desired to offer a novel and effective approach (e.g., an optimal delivery method and topical medication) for an ocular drug (e.g., a muscarinic acetylcholine receptor modulator) for preventing and / or treating ocular disorders, with improved efficacy, safety, and patient compliance, as compared with the traditional eye drop delivery route. Brief Summary of the Invention

[0011] The present invention provides an innovative approach for administering therapeutic agents, e.g., muscarinic acetylcholine receptor (mAChR) modulators, for preventing and / or treating ocular disorders through the eyelid. The eyelids, due to their close proximity to the conjunctiva - which is 2 to 30 times more permeable to drugs than the cornea - present a promising route for drug delivery. See N M Davies, Clin Exp Pharmacol Physiol. 2000 Jul;27(7):558-62. Since the conjunctiva directly interfaces with the eyeball and adjacent tissues, drugs reaching this tissue may effectively distribute to both anterior and posterior ocular regions, the main targets in many eye diseases. Transdermal delivery via the eyelid skin may offer advantages over conventional eye drops, including sustained drug levels at the site, fewer drawbacks, improved patient convenience (especially for chronic diseases like glaucoma), and the potential for drug delivery even during sleep, enhancing comfort, retention time, and overall quality of life.

[0012] Despite this potential, eyelid-based therapies are generally more effective for anterior segment eye diseases (e.g., involving the cornea, conjunctiva, iris, or aqueous humor), due to their direct exposure and fewer anatomical barriers. However, the eye’s natural defenses - tear film dilution, tight epithelial junctions, and nasolacrimal drainage - significantly limit drug absorption. As a result, only 1-7% of topically applied drugs typically reach the anterior chamber’s aqueous humor.

[0013] The posterior segment, which includes the retina and vitreous humor, is even more challenging to target with eyelid-delivered drugs. Barriers such as the blood-retinal barrier and the limited permeability of the sclera and choroid prevent effective drug delivery to these areas via topical administration. As a result, treatments for posterior segment diseases often require more invasive methods, such as intravitreal injections or systemic administration, to achieve a therapeutically effective drug concentration.

[0014] The present invention addresses this need by providing a novel approach to ocular medication delivery to posterior segments through the eyelid skin, offering a promising advancement in the treatment of posterior ocular diseases. Eyelid administration offers a more straightforward and less intimidating application process, which is especially beneficial for the elderly and parents administering medication to their children. This method solves the difficulties associated with eye drop applications, such as the risk of contamination or the challenge of keeping the eye open during application. This approach not only enhances patient compliance, but also reduces the physical and emotional stress often associated with eye drop administration, making it a more viable and convenient option for effective ocular treatment.

[0015] In this invention, innovative eyelid delivery systems combine therapeutic agents with pharmaceutical preparations for eyelid application. The drugs are mAChR modulators, specifically mAChR antagonists atropine and pirenzepine. All selected drugs can be designed and incorporated with emerging delivery systems and technologies, such as hydrogels, nano-micelles, liposomes, lipid-based nanoparticles, suspensions, emulsions, micro-needles, vesicles, and cyclodextrin complexes. These systems with drugs are then formulated into innovative preparations, including gels, hydrogels, ointments, creams, films, patches, gel patches, cataplasm, spraying solutions, liniments, wipes, film-forming solutions, gel forming solutions, or instilling eye drops which can be directly applied onto eyelids. The duration of drug release may range from a few seconds (e.g., with a single-use wipe) to up to 12 hours (e.g., with sustained-release patches) following a single daily or twice daily administration. It was discovered that via the eyelid skin application, drugs not only could avoid the contact with corneal surface, causing potential ocular irritation, but also had much lower drug exposure in anterior ocular tissues, such as fluid like aqueous humor and iris ciliary body (ICB), thereby reducing side effects such as accommodation and photophobia. Further, it was found that the present invention could effectively deliver drugs to the back of the eye (posterior segment of the eye) with sufficient pharmacological therapeutic levels to treat ocular disorders and less systemic absorption, which shows superior advantages to conventional approaches (e.g., eye drops and invasive injections).

[0016] In this invention, the mAChR modulators embrace the agonists, antagonists and positive allosteric modulators (PAMs), including, e.g., atropine or atropine sulfate, anisodamine, benzetimide, choline, dicyclomine, emraclidine, fesoterodine fumarate, homatropine, L- hyoscyamine, levamlodipine, levetiracetam, methscopolamine, nortropine, otenzepad, otilonium bromide, pilocarpine, pirenzepine or pirenzepine hydrochloride, pridinol methanesulfonate salt, scopolamine HBr trihydrate, sofpironium bromide, TBPB, telenzepine dihydrochloride, arecoline hydrobromide, guvacoline hydrochloride, tropicamide and cyclopentolate, TAK-071, MK-7622, VU0119498, BAY-2413555, Clozapine-d8, VU 0238429, VU0152100, VU0486846, VU0238441, VU6000918, VU10010, LY 2033298, xanomeline and any crystalline hydrates, acid salt forms, or isotopically labeled derivatives. Pirenzepine or pirenzepine hydrochloride, atropine or atropine sulfate, and any crystalline hydrates or acid salt forms thereof are preferably taken as the drugs for development for this invention.

[0017] Atropine sulfate, traditionally used for pupil dilation, at its lower concentration of 0.01% has gained approval in China, and recently in the European Union (EU), for controlling myopia in children. Its lower concentration of 0.025% was approved in Japan for controlling myopia in children. While atropine sulfate has been administered through eye drops into the eye, an eyelid administration method has not been previously reported. The M3 receptor's activation by atropine can lead to undesirable side effects such as mydriasis and photophobia, which are mainly caused by anterior iris and ciliary body tissues. The proposed eyelid delivery method aims to minimize atropine concentration in the anterior segment of the eye, ensuring that the drug effectively reaches the posterior segment for optimal efficacy while getting rid of the adverse effects.

[0018] Pirenzepine, with its extensive clinical history as a gastrointestinal medication in Europe and Asia, boasts an excellent safety profile. Although the exact anti-myopia target site in the eye remains under investigation, it is recognized that pirenzepine impacts neural structures predominantly located in the posterior segments. Previous animal studies have utilized intravitreal or subconjunctival injections to deliver pirenzepine to these posterior segments. See, e.g., E.M. Leech et al., Ophthalmic and Physiological Optics, 15(5), 351-356; C.L. Cottriall et al., Ophthalmic and Physiological Optics, 1999, 19(4), 327-335; D.L. Nickla et al., Experimental eye research, (2019) 181, 5-14; and L. Qian et al., Cell Biochemistry and Biophysics, 2015, 71(3), 1373-1378.

[0019] In human clinical studies, pirenzepine hydrochloride was formulated with hydroxypropyl methylcellulose (HPMC) to prepare a viscous hydrogel eye drop. However, due to pirenzepine hydrochloride's hydrophilic nature, corneal penetration and ocular bioavailability were suboptimal. See, e.g., Cottriall, C. L., & McBrien, N. A. (1996). The M1 muscarinic antagonist pirenzepine reduces myopia and eye enlargement in the tree shrew. D. Tan et al., Investigative Ophthalmology & Visual Science, 2005, 37(7), 1368-1379. One-year multicenter, double-masked, placebo- controlled, parallel safety and efficacy study of 2% pirenzepine ophthalmic gel in children with myopia. S.E. Olitsky, Ophthalmology, 2005, 112(1), 84-91; Safety and Efficacy of 2% Pirenzepine Ophthalmic Gel in Children With Myopia: A 1-Year, Multicenter, Double-Masked, Placebo- Controlled Parallel Study. Journal of Pediatric Ophthalmology and Strabismus, 42(1), 63; and R.M. Siatkowski et al., (2008). Two-year multicenter, randomized, double-masked, placebo- controlled, parallel safety and efficacy study of 2% pirenzepine ophthalmic gel in children with myopia. J. Amer. Association for Pediatric Ophthalmology and Strabismus, 12(4), 332-339.

[0020] The primary objective of this invention is to develop a series of ocular topical eyelid delivery systems for myopia prevention and treatment that incorporate pirenzepine’s intrinsic M1 / 4 selectivity. As such, this invention may achieve lower (than atropine) potential anterior adverse effects, combined with the eyelid route’s lower anterior exposure, to minimize or potentially eliminate anterior side effects.

[0021] To achieve more drugs effectively delivered to the back of the eye, the present invention incorporates the selected therapeutic reagents with emerging delivery systems and technologies, such as hydrogels, nano-micelles, liposomes, lipid-based nanoparticles, suspensions, emulsions, micro-needles, vesicles, and cyclodextrin complexes. These systems combined with drugs are finally formulated into innovative preparations, classifying as ophthalmic solutions (e.g., spray, aerosol, film-forming liquid, liniment, and eye drops), semi-solids (e.g., ointment, cream, gel, gel patch, cataplasm, and gel forming solution), and solid preparations (e.g., films, wipe, and patches), which can be easily and directly applied onto eyelids. By increasing the drug’s adhesion on eyelids, the preparations can promote delivery to posterior segment and prolong the retention of drugs.

[0022] Compared to traditional eye drops, this invention provides an optimal delivery approach for mAChR modulators, potentially revolutionizing the prevention or treatment of myopia and other ocular disorders in the posterior segment.

[0023] In one aspect, the prevention invention provides a method for ocular drug delivery, comprising a step of topically applying a therapeutically effective amount of a pharmaceutical composition to an eyelid skin of a subject, wherein the pharmaceutical composition comprises: (a) a therapeutically active agent comprising a muscarinic acetylcholine receptors (mAChRs) modulator; and (b) a pharmaceutically acceptable carrier.

[0024] In some embodiments, the muscarinic acetylcholine receptor modulator comprises atropine, pirenzepine, or a crystalline hydrate or salt thereof.

[0025] In some embodiments, the pharmaceutical composition comprises the muscarinic acetylcholine receptor modulator at a concentration ranging from about 0.01% (w / w) to about 10% (w / w). Preferably, the muscarinic acetylcholine receptor modulator is contained at a concentration ranging from about 0.01% (w / w) to about 8 % (w / w), or from about 0.1% (w / w) to about 6 % (w / w).

[0026] In some embodiments, the pharmaceutical composition comprises a penetration enhancer to facilitate the permeation of the muscarinic receptor modulator.

[0027] Examples of the penetration enhancer may include, but are not limited to, propylene glycol, polyethylene glycol (PEG) 200, polyethylene glycol 300, polyethylene glycol 400, hydroxypropyl- β-cyclodextrin (HP-β-CD), sulfobutyl ether-β-cyclodextrin (SBE-β-CD), ureido-β-cyclodextrin derivatives, hydroxypropyl-γ-cyclodextrin (HP-γ-CD), and γ-cyclodextrin (γ-CD). For instance, the penetration enhancer may include glycerin, polyethylene glycol, or propylene glycol, at a concentration ranging from about 0.01% (w / w) to about 98 % (w / w).

[0028] In some embodiments, the pharmaceutical composition comprises a surfactant.

[0029] Examples of the surfactant may include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, PEG-20 cetyl ether, PEG-35 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-30 stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, Pluronic F68, Pluronic F127, povidone, tocopherol polyethylene glycol succinate, benzalkonium bromide and benzalkonium chloride (BAK).

[0030] In some embodiments, the pharmaceutical composition comprises a gelling agent and / or a wetting agent.

[0031] Examples of the gelling agent or wetting agent may include, but are not limited to, glycerin, propylene glycol, polyethylene glycol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, deacetylated gellan gum, polyvinyl alcohol, carbomer, sodium hyaluronate, sodium oleate amide sulfonate, silicone oil, castor oil, white petrolatum, liquid petroleum, or lanolin.

[0032] In some embodiments, the concentration of the gelling agent and / or wetting ranges from about 0.1% (w / w) to about 20 % (w / w), or preferably from about 1.0% (w / w) to about 15% (w / w).

[0033] In some embodiments, the gelling agent may comprise hydroxypropyl methylcellulose or sodium hyaluronate, at a concentration ranging from about 0.01% (w / w) to about 10 % (w / w).

[0034] In some embodiments, the pharmaceutical composition comprises one or more thickening agents, osmotic pressure regulators, pH regulators, or preservatives.

[0035] Examples of the thickening agents include, but are not limited to, HPMC, CMC-Na, sodium hyaluronate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, microcrystalline cellulose, sodium carboxymethylcellulose, anhydrous glucose, and any combination thereof.

[0036] In some embodiments, the thickening agent is HPMC, sodium hyaluronate, CMC-Na, or any combination thereof.

[0037] In some embodiment, the concentration of the thickening agent ranges from about 0.1% (w / w) to about 5.0% (w / w), or preferably from about 0.1% (w / w) to about 1.00% (w / w).

[0038] Examples of the osmotic pressure regulator include, but are not limited to, mannitol, glucose, sorbitol, glycerol, polyethylene glycol, propylene glycol, and any combination thereof. The concentration of the osmotic pressure regulator may range from about 0.01% (w / w) to about 10.0% (w / w).

[0039] Examples of the pH regulator include, but are not limited to, tromethamine, sodium hydroxide, hydrochloric acid, and any combination thereof. The concentration of the pH regulator may range from about 0.01% (w / w) to about 5.0% (w / w).

[0040] In some embodiments, the pH of the pharmaceutical composition ranges from about 4.0 to about 10.0.

[0041] In some embodiments, the pharmaceutically acceptable carrier may include a gel, a hydrogel, an ointment, cream, film, patch, gel patch, cataplasm, spraying solution, aerosol, film-forming solution, gel forming solution, liniment, wipe, or eye drop solution.

[0042] In some embodiments, the pharmaceutical composition is directly and primarily applied to the eyelid skin.

[0043] In some embodiments, the pharmaceutical composition is directly and primarily applied to the eyelid skin, and covered with a film, a patch, or a gel patch. For instance, examples of the film or patch may include Tegaderm™, Mepitel®Film, Opsite®Gentle and other commercially available medical films or patches.

[0044] In some embodiments, the pharmaceutical composition is in a form selected from the group consisting of gels, ointments, creams, films, patches, gel patches, cataplasms, sprays, film-forming solutions, gel-forming solutions, liniments, wipes, and eye drops adapted for eyelid application.

[0045] In some embodiments, the duration of drug delivery of the pharmaceutical composition ranges from approximately a second to approximately up to twelve hours.

[0046] In some embodiments, the pharmaceutical composition is applied by wiping, brushing, spraying, or use of a sustained-release patch.

[0047] In some embodiments, the pharmaceutical composition is applied once or twice or multiple times a day.

[0048] In some embodiments, the method has a lower drug delivery to anterior segment and a lower systemic drug exposure, as compared to a method administrating the same amount of the muscarinic acetylcholine receptor modulator via eye drop administration into an eye.

[0049] In some embodiments, ocular drug delivery is used for prevention or treatment of an ocular disorder.

[0050] In some embodiments, the ocular disorder comprises myopia, or a posterior segment eye disease (e.g., posterior segment eye diseases including age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma causing optic nerve damage). For instance, the ocular disorder may be child or youth myopia (e.g., for kids under the age of 18).

[0051] As used herein, the term “or” may also imply “and,” unless such an interpretation results in a contradiction or infeasibility.

[0052] As used herein, the terms “treating,” “treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and / or therapy.

[0053] As used herein, the terms "individual," "patient," or "subject" are used interchangeably. None of the terms require or are limited to situations characterized by supervision (e.g. constant or intermittent) from a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker).

[0054] As used herein, a "therapeutically effective amount" refers to a sufficient amount of a muscarinic acetylcholine receptor (mAChRs) modulator, at a reasonable benefit / risk ratio applicable to treating ocular disorders in a subject in need thereof. It is understood, however, that the total daily usage of a muscarinic acetylcholine receptor (mAChRs) modulator may be decided by the attending physician or personal coach within the scope of sound medical judgment. The specific effective dose level for any particular subject will depend upon a variety of factors including the other disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration and route of administration; the duration of the administration; drugs used in combination or coincidental with a muscarinic acetylcholine receptor (mAChRs) modulator; and like factors well known in the medical arts or sports science. In addition, a "therapeutically effective amount" is the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician.

[0055] Those with ordinary skill in the art of preventing or treating ocular disorders recognize that an amount may be considered "effective" even if the condition is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject. Various indicators for determining the effectiveness of a method are known to those skilled in the art of preventing or treating ocular disorders in a subject in need thereof.

[0056] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. Other terms are defined herein within the description of the various aspects of the invention. Brief Descriptions of the Drawings

[0057] Fig. 1 illustrates skin permeation profiles of representative aqueous gel formulations in Example 2.

[0058] Fig. 2 illustrates skin permeation profiles of aqueous gel formulations in Example 3-F1 to F5.

[0059] Fig. 3 illustrates skin permeation profiles of aqueous gel formulations in Example 3-F6 to F12.

[0060] Fig. 4 illustrates skin permeation profiles of aqueous ophthalmic formulations in Example 1.

[0061] Fig.5 illustrates the comparison of the mean ocular tissue, and fluid concentrations of eyelid application (according to some embodiments of the present invention) vs. eye drops in Example 12.

[0062] Fig.6 illustrates the comparison of the mean ocular tissue, and fluid concentrations of eyelid application (according to some embodiments of the present invention) vs. eye drops in Example 13.

[0063] Fig. 7 illustrates minimal anterior segment exposure and negligible systemic absorption (according to some embodiments of the present invention) in Example 13. Detailed Description of the Invention

[0064] Reference will now be made in detail to the preferred embodiments of the invention, examples of which are further illustrated. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. To the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the claims. Furthermore, in the detailed description of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be obvious to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and other features have not been described in detail as not to unnecessarily obscure aspects of the present invention.

[0065] Generally speaking, the present invention provides a novel drug delivery system for muscarinic acetylcholine receptor (mAChR) modulators, administered via the eyelid, including atropine (or atropine sulfate), pirenzepine (or pirenzepine dihydrochloride), etc. This system allows for a controlled and / or sustained release of the drug, bypassing the rapid clearance typically associated with traditional eye drop administration methods. The carrier for the drug can be an aqueous formulation, such as hydrogel or polymer matrix, or non-aqueous formulation, all designed for adherence to the eyelid skin and controlled release of the drug. Atropine sulfate or pirenzepine dihydrochloride can be converted to their respective base forms and form salts with other organic acids, such as sorbic acid, maleic acid, fumaric acid, citric acid, etc., to enhance their hydrophobicity. The concentration of atropine sulfate or pirenzepine dihydrochloride in the formulation may vary from 0.001% to 10% (w / w), with a preference for concentrations ranging from 0.01% to 8%.

[0066] The invention is further elucidated with specific examples to illustrate the method. It is important to note that these examples serve merely to describe the invention and do not intend to limit its scope. Experiments will be conducted to enhance the chemical stability and improve the efficacy of delivery to the posterior eye segments for atropine sulfate and / or pirenzepine. Experimental methods without specified conditions are generally prepared under conventional conditions found in the literature or as suggested by the excipient manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or fractions in this invention are calculated on a weight- by-weight (w / w) basis. All professional and scientific terms used herein are assumed to be understood by well-trained personnel in the field. Additionally, any methods and materials similar or equivalent to those described in this invention can be applied. The preferred embodiments and materials described herein are used only for exemplary purposes. Example 1. Aqueous Formulations with muscarinic acetylcholine receptor modulators

[0067] The pirenzepine dihydrochloride has a wide range of solubility in water (high to 110 mg / ml) while its base is limited in solubilization to develop formulations. Therefore, to get higher solubilization, the solubility of pirenzepine base in water and different pharmaceutical reagents such as PEG 400 and glycerin was investigated.

[0068] The method for solubility test of pirenzepine base: Suitable amount of pirenzepine base was weighted to a 2 ml tube with cap, and then water, PBS or reagents such as PEG 400 and glycerin were added into the tube, and the base was dissolved via vortex to a dispersed suspension. In some samples, water was added to the pirenzepine base mixture to get a predetermined percentage of pharmaceutical solutions. All samples were filtered and the filtrates are diluted with 50% methanol to detect the solubilized amount of pirenzepine base.

[0069] As shown in Table 1, Pirenzepine base had relatively lower solubility in water compared to that in pure pharmaceutical reagents (PEG 400 and glycerin) or their 5% to 50% dilutions. From the perspective of dosage, 50% or pure PEG 400 and pure glycerin could solubilize at least 2% Pirenzepine base or 2.1% Pirenzepine base hydration which equaled to 2.52% pirenzepine dihydrochloride for preparation development. Contrarily, Pirenzepine base was barely solubilized in oily excipients such as White Petrolatum and Lanolin, indicating that aqueous formulations appear to be the priority choice for composition development. Table 1 Solubility test of Pirenzepine base in water and pharmaceutical reagents Pharmaceutical solvents and Solubility of Pirenzepine base Sam le H Example 1-S5 50% PEG400 7.8 20.08 Example 1-S6 5% PEG400 in buffer (8.9) 8.8 5.99 of as listed in Table 2. Table 2 Aqueous formulations with pirenzepine dihydrochloride or pirenzepine base Number of Pirenzepine Pirenzepine base Other components r r r Example 1-F9No 2.10% 50% PEG400, Purified waterExample 1-F10N 210% 979% PEG400 P rifi d w t runt ter under continuous stirring until completely dissolved. A measured quantity of glycerin was added to the pirenzepine dihydrochloride solution. The pH values of the pirenzepine dihydrochloride solution was adjusted to approximately 5.0-5.3 using 10 M sodium hydroxide or 1 M hydrochloric acid. Mannitol was optionally included to modulate the osmolarity to 200-3000 mOsmol / kg. Finally, purified water was added to achieve a total weight of 5.0 g. For Example 1-F7, a specified amount of pirenzepine base was accurately weighed and added to 1.5 g of glycerin under continuous stirring until completely dissolved. Osmolarity was adjusted to 200-3000 mOsmol / kg using mannitol if necessary. A total weight of 5.0 g aqueous preparation was supplemented with purified water. All formulations were prepared in accordance with the specified compositions outlined in this example. The pH and osmolarity of these formulations were measured. Example 2. Aqueous ophthalmic gel Formulations with muscarinic acetylcholine receptor modulators

[0072] To improve ocular bioavailability, formulations were developed incorporating penetration enhancers that were recognized for their safety and efficacy in ocular products. The refined formulation was designed to improve the permeability of the active pharmaceutical ingredient (API) across ocular barriers without inducing irritation.

[0073] The formulation included the API, a hydrogel base or ointment base, wetting / gelling agents, surfactants and penetration enhancers. The mAChR modulator, e.g., atropine sulfate, pirenzepine dihydrochloride, or their base form (or other suitable salt or crystalline hydrate forms), was uniformly dispersed within the gel or white petrolatum to ensure consistent distribution, as well as increasing the effectiveness of the formulations and comfort of subjects to be administered with the formulations. The API concentration was precisely adjusted to ensure both optimal efficacy and safety, e.g., ranging from 0.01% to 10% (w / w), preferably from 0.1% to 6% (w / w).

[0074] Wetting or gelling agents such as glycerin, propylene glycol, polyethylene glycol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, deacetylated gellan gum, polyvinyl alcohol, carbomer, sodium hyaluronate, sodium oleate amide sulfonate, silicone oil, castor oil, white petrolatum, liquid petroleum, and lanolin could be added to soften the gelling texture and keep the eyelid skin moisturized. To facilitate the API's passage across the ocular membrane, inventors incorporated a selection of various penetration enhancers. These might include glycerin, propylene glycol, polyethylene glycol (PEG) 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol, polypropylene glycol, hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutyl ether-β- cyclodextrin (SBE-β-CD), ureido-β-cyclodextrin derivatives, hydroxypropyl-γ-cyclodextrin (HP- γ-CD), and γ-cyclodextrin (γ-CD), which can enhance their solubility and bioavailability. The formulation could also contain surfactants including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, PEG-20 cetyl ether, PEG-35 castor oil, PEG-40 hydrogenated castor oil, PEG- 60 hydrogenated castor oil, PEG-30 stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, Pluronic F68, Pluronic F127, povidone, tocopherol polyethylene glycol succinate, benzalkonium bromide and benzalkonium chloride (BAK), etc.

[0075] In the formulations, these enhancers were used at optimized concentrations to promote drug permeation while ensuring the safety and comfort of the ocular surface. The inclusion of these enhancers was carefully balanced to achieve a synergistic effect, enhancing the overall permeability of the ocular tissue and allowing for more effective delivery of the API to the target site within the eye. The final formulation was tested for stability, pH, and viscosity, ensuring its suitability for ocular administration. The optimized formulation was intended for application to the eyelid or direct application onto the ocular surface, ensuring effective delivery of the API to the target tissues within the eye.

[0076] A series of aqueous ophthalmic gel formulations were prepared. These formulations contained varying concentrations of pirenzepine dihydrochloride (ranging from 0.01% to 8.0%) with HPMC (K100M or E4M) or sodium hyaluronate (0.5%~10%). A subset of these samples was selected for further studies, and their compositions are detailed in Table 3. Note 2.52% of pirenzepine dihydrochloride is equivalent to 2.0% of pirenzepine; 5.04% of pirenzepine dihydrochloride is equivalent to 4.0% of pirenzepine; and 7.56% of pirenzepine dihydrochloride is equivalent to 6.0% of pirenzepine. Table 3 Aqueous ophthalmic gel Formulation Compositions of Example 2-F1 to F30 Number of Pirenzepine G lli t Oth t 0.01~0.5% Sodium acetate, 0.001~2% BAK, 0.015% EDTA·2Na, NaOH and Hydrochloride, Purified water 0.01~0.5% Sodium acetate, Example 2- 0.001~2% BAK, 0.015% EDTA·2Na, NaOH and Hydrochloride, Purified water in this example. For instance, for pirenzepine dihydrochloride formulations containing HPMC (Examples 2-F1 to F21): a specified amount of pirenzepine dihydrochloride was accurately weighed and slowly added to 1.5 g of purified water under continuous stirring until completely dissolved. A measured quantity of HPMC K100M was dissolved in purified water to create a concentrated gel. Sodium acetate, benzalkonium chloride (BAK), and disodium edetate were dissolved in purified water to prepare an inorganic salt solution. A specific volume of this inorganic salt solution was added to the pirenzepine dihydrochloride solution. The pH values of both the pirenzepine dihydrochloride solution and the HPMC gel were adjusted to approximately 5.0-5.3 using 10 M sodium hydroxide or 1 M hydrochloric acid. Subsequently, the HPMC gel was slowly added to the pirenzepine dihydrochloride solution under continuous stirring. Sodium chloride or mannitol was optionally included to modulate the osmolarity to 200-1000 mOsmol / kg. Finally, purified water was added to achieve a total weight of 5.0 g. All formulations were prepared in accordance with the specified compositions outlined in this example. The pH, osmolarity, and average viscosity of formulations Example 2-F1 to F30 were measured. Viscosity was assessed using a DHR-2 rheometer (TA Instruments) under a shear rate of 1 s⁻¹ at 20°C. Example 3. Aqueous gel Formulations with wetting agents

[0078] Based on the gel preparations in Example 2, to keep the moisturization and continual drug diffusion of the applied formulations on eyelids, wetting agents including glycerin, propylene glycol, or Polyethylene Glycol (PEG) are added to the aqueous ophthalmic gel formulations. A subset of these samples was selected for further studies, and their compositions are detailed in Table 4. Table 4 Aqueous gel Formulation Compositions with wetting agents of Example 3-F1 to F13 Number Pirenzepine Wetting 0.01~0.5% Sodium acetate, 15% Example 1.62% HPMC 0.001~2% BAK, 0.015% EDTA·2Na, NaOH and Hydrochloride, Purified water ed compositions outlined in this example. For example, for pirenzepine dihydrochloride formulations containing HPMC (Examples 3-F1 to F13): a specified amount of pirenzepine dihydrochloride was accurately weighed and slowly added to 1.5 g of purified water under continuous stirring until completely dissolved. A measured quantity of HPMC K100M was dissolved in purified water to create a concentrated gel. Sodium acetate, benzalkonium chloride (BAK), and disodium edetate were dissolved in purified water to prepare an inorganic salt solution. A specific volume of this inorganic salt solution was added to the pirenzepine dihydrochloride solution. Then, glycerin or Polyethylene Glycol 400 were added according to the specific formulations to the pirenzepine dihydrochloride solutions. The pH values of both the pirenzepine dihydrochloride solution and the HPMC gel were adjusted to approximately 5.0-5.3 using 10 M sodium hydroxide or 1 M hydrochloric acid. Subsequently, the HPMC gel was slowly added to the pirenzepine dihydrochloride solution under continuous stirring. Sodium chloride or mannitol was optionally included to modulate the osmolarity to 200-1000 mOsmol / kg. Finally, purified water was added to achieve a total weight of 5.0 g. All formulations were prepared in accordance with the specified compositions outlined in this example. The pH, osmolarity, and average viscosity of the formulations were measured. Viscosity was assessed using a DHR-2 rheometer (TA Instruments) under a shear rate of 1 s⁻¹ at 20°C. Example 4. Ointment based Ophthalmic Formulations

[0080] Ointment formulations can form a physical barrier covering the skin surface, reduce water evaporation and isolate the eyelids from external stimuli. The ointment formulations containing Pirenzepine base or Pirenzepine dihydrochloride, oily base, lubricants or wetting agents were prepared. Table 5 detailed a subset of these samples and their compositions selected for further studies. Table 5 Ointment based Formulation Compositions of Example 4-F1 to F6 Number of Ointment API Wetting agents Other components l 0.4% Example 55% White Pirenzepine 18% Lanolin Purified water ed on containing White Petrolatum (Examples 4-F1) as an example: a specified amount of pirenzepine dihydrochloride was accurately weighed and slowly added to 0.15 g of Liquid petroleum under continuous stirring until completely dispersed. A measured quantity of melted White Petrolatum was added to the pirenzepine dihydrochloride dispersion to achieve a total weight of 5.0 g.

[0082] For both Examples 4-F3 and Examples 4-F6: a specified amount of pirenzepine dihydrochloride (or pirenzepine base) was accurately weighed and slowly added to purified water under continuous stirring. The pH value the pirenzepine dihydrochloride solution was adjusted to approximately 5.0-5.3 using 10 M sodium hydroxide or 1 M hydrochloric acid. For pirenzepine base, there is no need to adjust the pH, thus obtaining an alkaline solution. The pirenzepine dihydrochloride (or pirenzepine base) solution was then dispersed to a measured quantity of Lanolin. Finally, the melted white Petrolatum was slowly added to the pirenzepine dihydrochloride solution to a total weight of 5.0 g under continuous stirring.

[0083] All formulations were prepared in accordance with the specified compositions outlined in this example. The pH and average viscosity of formulations Example 4-F1 to F6 were measured. Viscosity was assessed using a DHR-2 rheometer (TA Instruments) under a shear rate of 1 s⁻¹ at 20°C. Example 5. Method of Eyelid Application

[0084] According to the present invention, the application of an aqueous gel formulation or non- aqueous ointment formulation to the eyelid is easy. In some embodiments, to apply the formulation to the upper eyelid, a designated quantity could be placed on the fingertip. Using a gentle, precise motion, the formulation could be swiped along the upper eyelid skin, starting from the inner canthus and proceeding towards the outer canthus. This could ensure even distribution and contact with the ocular surface. Similarly, for the lower eyelid, the application could follow an inverse path. The fingertip, bearing the formulation, could glid along the lower eyelid margin, but in the opposite direction – from the outer canthus to the inner canthus. This method could be repeated for both eyelids to ensure thorough coverage and absorption.

[0085] The exact amount of formulation applied was critical, as it was determined by the specific surface area of the eyelid and the desired API concentration. This personalized approach could guarantee that the application was both effective and comfortable for the user, maximizing the bioavailability of the drug while minimizing waste.

[0086] The application of an aqueous gel formulation or non-aqueous ointment formulation to the eyelid can be covered with a film, a patch or gel patch to provide enhanced drug retention, water loss prevention, and / or controlled delivery of the active drug agent.

[0087] The film or patch may comprise Tegaderm™, Mepitel®Film, Opsite®Gentle and other commercially available medical films or patches. The film or patch is selected for its flexibility, and moisture vapor permeability, allowing the film or patch to conform comfortably to the skin while maintaining an appropriate barrier function.

[0088] Similarly, drug delivery systems such as films, cataplasm, patches, and gel patches could be directly applied to the eyelids. The duration of drug release might range from seconds (e.g., with a single-use wipe) to up to 12 hours (e.g., with sustained-release patches) following a once daily or twice daily administration. This method of eyelid application was designed to be straightforward yet precise, allowing for consistent and replicate results across various patient populations. It might leverage the unique properties of the gel or ointment formulations to provide a controlled and sustained release of the drug, directly targeting the posterior ocular tissues. Example 6. Skin permeation measurements of API in aqueous gel ophthalmic formulations

[0089] As can be seen from the in vivo ocular pharmacokinetic results in Example 12 and Example 13, the eyedrop with 2% pirenzepine and HPMC K100M, one preparation from the patent WO / 02096418A1, which was studied in the clinical trial for topical installation use, showed effective drug distribution in retina / choroid. However, after applying it and its similar preparation with 6% pirenzepine (Example 2-F2 and F10) on eyelids, they both got significantly beyond or close to the high retinal / choroidal concentrations obtained by eyedrop administration. In other words, Example 2-F2 and F10 could be taken as reliable control samples for formulation development for eyelid application. Corresponding to the in vivo eyelid model, the classical method in vitro permeability test (IVPT) was built for evaluating drugs permeation through the skin, which to the great extent, can mimic the drug delivery into eyelid.

[0090] This example details the IVPT methodology for assessing the skin permeation of exemplary formulations containing mAChR modulators, administered via the eyelid, including atropine sulfate, pirenzepine dihydrochloride and their crystalline hydrate and salt forms.

[0091] The in vitro permeability test utilized Franz diffusion cells to measure the permeation of active ingredients across the skin. Excised Bama pig skin samples (with average thickness of 1 mm), ensuring the integrity and viability of the skin as a barrier, were fitted into the diffusion cells (volume: 18 ml). The donor compartment of the diffusion cell was loaded with a predetermined amount of the formulation, ensuring uniform distribution. The receptor compartment, filled with isotonic phosphate-buffered saline (PBS), was maintained at a controlled temperature of 32°C± 1 ℃ and stirring rate of 600 rpm. At regular intervals, 1 ml aliquots were collected from the receptor compartment, and an equal volume of fresh buffer was replenished to maintain sink conditions. The collected samples were analyzed using high-performance liquid chromatography (HPLC) to quantify the amount of drug permeated through the skin.

[0092] The cumulative amount of drug permeated versus time was plotted, and key pharmacokinetic parameters such as the permeability coefficient (Papp, cm / s) and the cumulative amount of drug permeated through the skin were calculated. These parameters provided insights into the efficiency of drug delivery and the potential therapeutic efficacy of the formulations.

[0093] To investigate the in vitro and in vivo correlation of aqueous gel formulations, the skin permeation test of aqueous gel formulations in Example 2 was executed by the IVPT methods referring to the ocular pharmacokinetic study in Example 12. Representative samples Example 2- F2 and F10 were subjected to Franz cell permeation tests, with results presented in Table 6 and Fig. 1. The findings revealed that the gel formulation Example 2-F2 exhibited less permeation compared to both Example 2-F2 and Example 2-F10 covered with Tegaderm film on the sample applied on skin surface. Here, the film can reduce the water evaporation in the applied sample to avoid it to be dried out. Example 2-F2 and F10 showed strong drug dose-response relationship in skin permeation with ascending administration, essentially consistent to the ocular distribution of pirenzepine by eyelid application. Therefore, these IVPT results can be the gold standard to quickly screen formulations developed in the invention. To put it more directly, those formulations accumulating drug amount far more than or approximate to the level of Example 2-F2 with film can be outlined for further studies. Table 6 Skin permeation study of aqueous gel formulations in Example 2 Osmolarity Viscosit Cumulative amount of permeated drug PappSam le H (mOsmol / (μm / cm2) s) 6 -80 -87-7

[0094] It is known that gelling agents become dry after applied on the skin instantly in one or a few hours, thus obstructing drugs from permeating the skin from the gel forms. To resolve the problem, Example 3 provides aqueous gel formulations introduced with wetting agents to enhance the moisturization of some aqueous gel formulations described in Example 2. Several aqueous gel formulations containing HPMC K100M as the gelling agent and PEG 400 as a wetting agent were prepared and then used for IVPT study by methods in Example 6.

[0095] The pirenzepine permeation profiles of Example 3-F1 to F3 were examined and the results are presented in Table 7 and Figure 2. Formulations in absence of wetting agents including Example 2-F2 and F10 served as control samples shown in Example 6. The permeation profiles demonstrated that Example 3-F1 and F2 were significantly higher in permeated drug amounts and appearance permeation parameters than those control formulations, despite their comparable viscosities. Formulations Example 3-F1 and F2 with lower API content even exceeded the control Example 2-F10 with three times higher API content in drug permeation. Comparably, the results also noted that Example 3-F3 still had slightly higher drug permeation than Example 2-F2 with no wetting agent even though its viscosity is lower. This pronounced difference highlights the superior skin permeating performance of PEG 400 in aqueous gel formulations compared with HPMC K100M. Table 7 Skin permeation study of aqueous gel formulations in Example 3-F1 to F5 Osmolarity Cumulative amount of the permeated Viscosity P (mOsmol / μ2appSamples pH drug ( g / cm ) ) or 3- F4 and F5, essentially permeated very low amounts of pirenzepine through the skin. This indicated that aqueous ophthalmic gel formulations taking PEG 400 as a wetting agent have more superior composition effect with HPMC K100M than other gelling agents such as Hydroxyethyl cellulose or Deacetylated gellan gum in skin permeation of drug. Besides, PEG 400 helps to keep the gel formulations with HPMC K100M moisturized and soft after application on the skin.

[0097] The findings from Example 6 and this example confirm that aqueous ophthalmic gel formulations containing HPMC K100M and PEG 400 exhibit excellent skin permeating properties for pirenzepine dihydrochloride compared to the control formulations with no PEG 400, which revealed effective drug distribution by upper eyelid administration (Example 13). In addition, regardless of whether their viscosities are as low as 20,000 mPa·s or as high as 60,000 mPa·s, the composites of HPMC K100M and PEG 400 can provide promising amount of drug to permeate the skin for efficacy. This discovery underscores the innovative value of the present invention in optimizing ophthalmic gel formulations for eyelid application. Example 8. Skin permeation of aqueous gel formulations with sodium hyaluronate and wetting agent

[0098] Apart from the PEG used for composition in Example 7, glycerin, a common humectant to formulate aqueous gels, provides wetness for gel products and skin, and contributes to drug permeation through the skin. This example prepared aqueous gel formulations containing pirenzepine dihydrochloride and gelling carriers combining glycerin with either sodium hyaluronate or HPMC K100M.

[0099] To achieve superior drug permeated through skin and eyelid application, moderately high viscosity is desired. This example selected formulations including Example 3-F2 to F6 from Example 3 for IVPT study. The viscosity of formulation Example 3-F1 was approximate to that of Example 2-F2 and F10, while formulations Example 3-F2 to F6 were adjusted to the viscosity ranging from 3,000 mPa·s to 70,000 mPa·s. Detailed composition characterizations and skin permeation results are presented in Table 8 and Figure 3. Table 8 Skin permeation study of aqueous gel formulations in Example 3-F6 to F12 Osmolarity Cumulative amount of the permeated Viscosity P l2apps) 2 9 5 7 2 8 5 8 2 8 pine dihydrochloride permeation than the control Example 2-F2, which contained no wetting agent, and Example 3-F1, which consisted of PEG 400 as the wetting agent. Among the serial formulation containing sodium hyaluronate and glycerin, the drug permeation of Example 3-F7 was notably higher than the controls Example 2-F2 and F10 tested in Example 6. These results suggest that glycerin surprisingly generates positive effect for sodium hyaluronate on drug permeation, but negative for HPMC K100M. Meanwhile, glycerin can maintain the wetness of the skin and gel formulations with sodium hyaluronate for continuous drug permeation.

[0101] This example has confirmed that the gel formulation combined with sodium hyaluronate and glycerin exceeded the transdermal pirenzepine level of the control samples which shows effective drug distribution in the posterior segment of eyes applied by upper eyelid. Moreover, this gel formulation can improve the moisturization of gel and skin, indicating great advantages in administration via eyelid. Taken together, this example provided sodium hyaluronate composited with glycerin as another prospective gel transdermal system option for eyelid application. Example 9. Skin permeation study of API in aqueous ophthalmic formulations

[0102] This example prepared several aqueous formulations containing pirenzepine dihydrochloride and pirenzepine base with or without wetting agents or penetrating enhancers for comparison, and then evaluated their skin permeation by IVPT methods as shown in Example 6.

[0103] The Franz permeation profiles of aqueous formulations Example 1-F1 to F9 were evaluated, with results presented in Table 9 and Figure 4. Compared to the aqueous solution of pirenzepine dihydrochloride (Example 1-F1), Example 1-F5 with pirenzepine base demonstrated significantly more permeated drug amount and higher Papp(1.288×10-7). Further analysis revealed that more drug permeation observed in Example 1-F2 and F3, compared to Example 1-F6 and F7, even though the Papp parameters from latter formulations were higher. When compared to the control and results in Example 6, Example 1-F7 achieved similar drug permeation with significant lower pirenzepine base content, demonstrating that aqueous formulation with pirenzepine base can also perform similar effects on eyelids as the control Example 2-F2. Table 9 Skin permeation study of aqueous ophthalmic formulations in Example 1 Osmolarity Cumulative amount of the permeated drug s) 0.5± 0.8± Example 1-F1 5.0 1931.8± ND ND ND0.07 0.10 3.596×10-9-8-8-8-7-7-7-8-8ite both being prepared in 15% glycerin solution, indicating that glycerin helps to permeate the pirenzepine dihydrochloride through skin but not the pirenzepine base. On the other hand, Example 1-F9 containing 15% PEG400 had an increased pirenzepine base permeation through the skin than Example 1-F8 and the control Example 2-F2 (in Example 6), showing that PEG400 is more favorable for the pirenzepine base than glycerin. The results suggest that the skin permeation of pirenzepine dihydrochloride or pirenzepine base is weak in some aqueous formulations, but it can be improved by adding the permeation enhancers such as glycerin and PEG400, which also have the capability to keep the samples continuously wet on the skin.

[0105] Different from the high viscosity of gel formulations, aqueous formulations have low viscosity and easily flow on skin, but they can avoid the formulations being dried out by adding a wetting agent. Aqueous formulations can be processed to be various dosage forms such as liniment, spray, and wipe, and filled in suitable containers to control the flowability in use. In this example, the prepared aqueous ophthalmic formulations were relatively more flowable compared to the sticky gel formulations, some of which still surpassed the control in IVPT tests, particularly the pirenzepine base formulation (Example 1-F7) with much lower API content.

[0106] Accordingly, the aqueous ophthalmic formulations containing pirenzepine dihydrochloride or pirenzepine base essentially take satisfactory effects in contrast to the control. The addition of wetting agents or penetrating enhancers can make contributions to drug permeation for aqueous ophthalmic formulations. For convenient use by applying on eyelids, the aqueous ophthalmic formulations can be processed into forms including liniment, spray, foam, and wipe in this invention. Example 10. Skin permeation study of Ointment based Formulation Compositions

[0107] In this example, ointment formulations Example 4-F3 and F6 were selected for IVPT test, and the results were shown in Table 10. The ointment formulations containing pirenzepine base or pirenzepine dihydrochloride permeated essentially no detectable drug through the skin, both worse than aqueous gel formulations (Example 6, 7, and 8) and the aqueous formulations (Table 9 in Example 9). Therefore, ointment formulations revealed less advantages than those aqueous formulations and there is a need in formulation optimizations for further studies. Table 10 Skin permeation study of Ointment based Formulation Compositions Osmolarity Cumulative amount of the permeated Viscosity Pps)

[0108] This example provides a series of formulations including atropine and atropine sulfate or their alternate forms (or other suitable salt or crystalline hydrate forms) as the APIs, a hydrogel base or ointment base, gelling agents, wetting agents, surfactants and penetration enhancers, pH adjuster, metal chelating agents, and osmolarity modulators. The formulation compositions were detailed in Table 11. Table 11 Formulation compositions with atropine and sulfate atropine for eyelid application Number of API Gelling agents Other components Sam les e, m m 0.01~2% EDTA·2Na, Mannitol, NaCl, Purified water e, m m Hydrochloride, Mannitol, NaCl, Purified water ed der continuous stirring until completely dissolved. Sodium acetate, benzalkonium chloride (BAK), and disodium edetate were dissolved in purified water to prepare an inorganic salt solution. A specific volume of this inorganic salt solution was added to the atropine solution. A measured quantity of PEG or glycerin was added to the pirenzepine dihydrochloride solution according to the formulations. A measured quantity of HPMC or sodium hyaluronate was dissolved in purified water to create a concentrated gel solution. Then, add an appropriate percentage of HPMC or sodium hyaluronate gel stock solution to the atropine solution according to the formulations. Mannitol or NaCl was optionally included to modulate the osmolarity to 200-3000 mOsmol / kg. Finally, purified water was added to achieve a total weight of 5.0 g.

[0110] Similar methods for Example 11-F7 to F12, a specified amount of sulfate atropine was accurately weighed and slowly added to 1.5 g of purified water under continuous stirring until completely dissolved. The pH value of the pirenzepine dihydrochloride solution was adjusted to approximately 4.0-6.0 using sodium hydroxide or hydrochloric acid solutions. A specific volume of this inorganic salt solution (containing sodium acetate, benzalkonium chloride (BAK), and disodium edetate) was added to the atropine solution. According to the formulations, after a measured quantity of PEG or glycerin was added to the pirenzepine dihydrochloride solution, add an appropriate percentage of HPMC or sodium hyaluronate gel stock solution to the atropine solution. Mannitol or NaCl was optionally included to modulate the osmolarity to 200-3000 mOsmol / kg. Finally, purified water was added to achieve a total weight of 5.0 g. Example 12. Stability evaluation

[0111] This example presents the concise stability evaluation process for the formulations, verifying the drug's integrity and efficacy over an extended period. The formulations were stored at different temperatures and humidity levels, and their physical and chemical properties were monitored over a period of 6 months. The assessment included physical and chemical attributes such as appearance, API content, impurities, pH, and viscosity to confirm that the formulations remained stable and effective. The specifications for quality control encompass appearance, assay, impurities, pH, viscosity, etc. The comprehensive data obtained would guide the establishment of appropriate shelf-life and storage guidelines, ensuring that such formulations meet high standards of safety, efficacy, and quality throughout their entire duration of use. Example 13. Pilot ocular pharmacokinetic and tissue distribution study

[0112] A pilot ocular pharmacokinetic and tissues distribution study was conducted to evaluate the bioavailability of 2% pirenzepine gel formulations applied to the eyelid skin versus eye drops administered via topical instillation. Male New Zealand White rabbits underwent a single-dose ocular pharmacokinetic tissue distribution assessment. A calibrated positive displacement pipette was used to administer 40 μL of 2% pirenzepine gel total into the lower conjunctiva pocket of each eye for eye drop administration. The upper eyelid skin was clipped the day before. Approximately 80 μL of the same formulation of 2% pirenzepine gel was dispensed on the eyelid skin. Care was taken to ensure the gel evenly smeared on the eyelid skin surface.

[0113] The animals were observed for any abnormal observations prior to euthanasia. Body weights were measured and recorded prior to euthanasia. Aqueous humor, iris ciliary body, retina, choroid, and sclera were collected from all animals at approximately 1-, 2-, and 4-hours post dose on Day 1. Ocular tissues and fluids were analyzed via liquid chromatography – tandem mass spectrometry (LC-MS / MS).

[0114] The pirenzepine concentration in aqueous humor, iris-ciliary body, sclera and retina / choroid after single topical eye drop and eyelid administration at 1hr, 2hr, and 4hr post dose, and total area under curve (AUC) are shown in Table 12. The comparison figures are shown in Fig. 5 which illustrates the comparison of the mean ocular tissue and fluid concentrations via eyelid (dash line) vs. eye drop (solid line).

[0115] The results indicated that this transdermal route resulted in lower drug concentrations in the posterior segment tissues, such as the retina, choroid, and sclera, compared to traditional eye drop administration. Additionally, drug levels in the aqueous humor and iris-ciliary body were significantly reduced with eyelid skin application. These results suggested that delivering pirenzepine through the eyelid skin might bypass anterior segment tissues, potentially offering a more direct and efficient pathway to the posterior segment of the eye. This finding was unexpected, as muscarinic acetylcholine receptor antagonists—such as atropine and tropicamide—commonly would induce ocular side effects like mydriasis and cycloplegia by inhibiting M3 receptors in the iris sphincter and ciliary body. These effects were typically observed with standard eye drop administration. However, delivering the drug via the eyelid skin might effectively bypass the anterior ocular tissues, and reduce or even eliminate associated adverse effects. Table 12 Mean concentration and AUC of pirenzepine in aqueous humor, iris-ciliary body, sclera and retina / choroid after single eye administration to New Zealand White rabbits AUC (ng*h / ml Tissue Formulation Mean Conc. (ng / ml or ng / g) or ng*h / g) Example 14. Confirmatory ocular pharmacokinetic and tissue distribution study

[0116] An ocular pharmacokinetic study was conducted to further evaluate the finding from the pilot study. Testing groups included 2% pirenzepine in HPMC K100M gel (Example 2-F2) applied by both eye drop and eyelid, and 6% pirenzepine gel with HPMC K100M (Example 2-F10) by eyelid. Each test article was instilled into a group of six rabbits weighing 1.5 to 2.8 kg. 40 microliters of test article were instilled into both eyes of the rabbits while 80 microliters of the samples were applied onto the upper eyelids of the rabbits and covered with Tegaderm film. The aqueous humor and vitreous humor were withdrawn using a syringe at 1, 2 and 4 hours after single instillation. For each timepoint, samples were collected from 4 eyes. The concentration of pirenzepine in plasma, aqueous humor, iris-ciliary body, sclera and retina / choroid was analyzed by LC-MS / MS technique. The concentration of pirenzepine in vitreous humor was below the quantification limit (10ng / ml). The mean concentration of pirenzepine at different timepoints in aqueous humor, sclera and retina / choroid are presented in the following Table 13. Comparison figures of drug concentrations are displayed in Fig. 6 which shows the comparison of the mean ocular tissue, and fluid concentrations of eyelid (dash line) vs. eye drops (solid line). Table 13 Mean concentration of pirenzepine in plasma, aqueous humor, iris-ciliary body, sclera and retina / choroid at different timepoint after single topical administration to New Zealand White rabbits Timepoint 1 h 2 h 4 h Concentration (Plasma Aqueous Humor: ng / mL,

[0117] The results clearly demonstrated that the anterior ocular tissues’ pirenzepine concentration was significantly lower in eyelid skin administration route than traditional eye drops, which confirmed the finding in pilot ocular pharmacokinetic study. For posterior ocular tissues, the eyelid route demonstrated higher concentrations at 2- and 4-hours post-administration than in 1-hour, reflecting its sustained release capability. The eyelid route exhibited lower concentrations of pirenzepine in the retina / choroid, and sclera compared to eye drops at the same dose level. However, HPMC 100M gel formulation with 6% pirenzepine exhibited significant improvement in drug delivery compared to a formulation with 2% pirenzepine, suggesting that therapeutic drug level to the posterior ocular tissue was achievable by eyelid skin dosing route. This was an unexpected breakthrough in ocular drug delivery systems.

[0118] Total pirenzepine exposure in the ocular tissues, fluid, and plasma is shown in Table 14 and Fig. 7. Specifically, Fig. 7 shows minimal anterior segment exposure and negligible systemic absorption. Table 14 The area under curve of pirenzepine in AH, ICB, retina / choroid, sclera, and plasma of 2%, 6% PIR Eyelid vs.2% PIR Eyedrop AUC (ng*h / ml Aqueous Iris-ciliary *h / ) H b d R ti / Ch id S l Pl [ ] e rug exposure n e an er or segmen was s gn can y ower compare o e opical instillation of eyedrop, and a relatively lower concentration was found in plasma by the eyelid, indicating a lower systemic presence by using eyelid application. These results implied that anterior segment side effects like pupil dilation and accommodation impairment, as well as systemic safety caused by eyedrops, would be much less a concern in eyelid skin delivery route.

[0120] In summary, eyelid skin as a novel route for ocular drug delivery has shown promise in delivering drugs effectively to the posterior segment of the eye, achieving therapeutic concentrations while minimizing exposure to anterior ocular tissues and systemic circulation. Such targeted delivery could potentially reduce side effects like pupil dilation and photophobia.

[0121] Studies indicated that the eyelid skin possesses a thinner stratum corneum and lower impedance compared to other skin areas, facilitating higher drug permeation. This characteristic makes it a valuable site for ophthalmic drug administration, potentially enhancing drug delivery to ocular tissues. Furthermore, topical drug application to the eyelid skin may bypass some of the anatomical and physiological barriers that typically hinder drug penetration to the posterior eye segment. This approach could offer a non-invasive alternative to traditional methods, improving patient compliance and reducing systemic side effects.

[0122] Utilizing the eyelid skin as an administration route, according to the present invention, presents a promising strategy for targeted ocular drug delivery, potentially enhancing therapeutic outcomes while minimizing adverse effects.

[0123] Although specific embodiments and examples of this invention have been illustrated herein, it will be appreciated by those skilled in the art that any modifications and variations can be made without departing from the spirit of the invention. The examples and illustrations above are not intended to limit the scope of this invention. Any combination of embodiments of this invention, along with any obvious extensions or analogs, are within the scope of this invention. Further, it is intended that this invention encompasses any arrangement, which is calculated to achieve that same purpose, and all such variations and modifications fall within the scope of the appended claims.

[0124] All the features disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example of a generic series of equivalent or similar features.

Claims

WHAT IS CLAIMED IS:

1. A method for ocular drug delivery, comprising a step of topically applying a therapeutically effective amount of a pharmaceutical composition to an eyelid skin of a subject, wherein the pharmaceutical composition comprises: (a) a therapeutically active agent comprising a muscarinic acetylcholine receptor (mAChRs) modulator; and (b) a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the muscarinic acetylcholine receptor modulator comprises atropine, pirenzepine, or a crystalline hydrate or salt thereof.

3. The method of claim 1 or 2, wherein the muscarinic acetylcholine receptor modulator comprises pirenzepine or pirenzepine hydrochloride, or atropine or atropine sulfate.

4. The method of any one of claims 1 to 3, wherein the pharmaceutical composition comprises the muscarinic acetylcholine receptor modulator at a concentration ranging from about 0.01% (w / w) to about 10% (w / w).

5. The method of claim 4, wherein the pharmaceutical composition comprises the muscarinic acetylcholine receptor modulator at a concentration ranging from about 0.01% (w / w) to about 8 % (w / w) or from about 0.01% (w / w) to about 6 % (w / w).

6. The method of any one of claims 1 to 5, wherein the pharmaceutical composition further comprises a penetration enhancer to facilitate the permeation of the muscarinic receptor modulator through the eyelid skin.

7. The method of claim 6, wherein the penetration enhancer comprises glycerin, propylene glycol, polyethylene glycol (PEG) 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol, polypropylene glycol, hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutyl ether-β-cyclodextrin (SBE-β-CD), ureido-β-cyclodextrin derivatives, hydroxypropyl-γ-cyclodextrin (HP-γ-CD), or γ-cyclodextrin (γ-CD).

8. The method of claim 7, wherein the penetration enhancer comprises glycerin, polyethylene glycol or propylene glycol, at a concentration ranging from about 0.01% (w / w) to about 98 % (w / w).

9. The method of any one of claims 1 to 8, wherein the pharmaceutical composition further comprises a surfactant.

10. The method of claim 9, wherein the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, PEG-20 cetyl ether, PEG-35 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-30 stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, Pluronic F68, Pluronic F127, povidone, tocopherol polyethylene glycol succinate, benzalkonium bromide or benzalkonium chloride (BAK).

11. The method of any one of claims 1 to 10, wherein the pharmaceutical composition further comprises a gelling agent and / or a wetting agent.

12. The method of claim 11, wherein the gelling agent and / or a wetting agent comprises glycerin, propylene glycol, polyethylene glycol, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, deacetylated gellan gum, polyvinyl alcohol, carbomer, sodium hyaluronate, sodium oleate amide sulfonate, silicone oil, castor oil, white petrolatum, liquid petroleum, or lanolin.

13. The method of claim 12, wherein the gelling agent comprises hydroxypropyl methylcellulose or sodium hyaluronate, at a concentration ranging from about 0.01% (w / w) to about 10 % (w / w).

14. The method of any one of claims 1 to 13, wherein the pharmaceutical composition further comprises a thickening agent, an osmotic pressure regulator, a pH regulator, a buffer regulator, a preservative, a film-forming agent, a propellant, a tissue material, or a backing material.

15. The method of any one of claims 1 to 14, wherein the pharmaceutically acceptable carrier comprises a gel, hydrogel, ointment, cream, film, patch, gel patch, spraying solution, aerosol, film-forming solution, gel forming solution, or eye drop solution.

16. The method of any one of claims 1 to 15, wherein the pharmaceutical composition is directly and primarily applied to the eyelid skin.

17. The method of any one of claims 1 to 16, wherein the pharmaceutical composition is directly and primarily applied to the eyelid skin, and covered with a film, a patch, or a gel patch.

18. The method of any one of claims 1 to 17, wherein the pharmaceutical composition is in a form selected from the group consisting of gel, hydrogel, ointment, cream, film, patch, gel patch, cataplasm, spray, liniment, wipe, film-forming solution, gel-forming solution, and eye drop adapted for eyelid application.

19. The method of any one of claims 1 to 18, wherein the duration of drug delivery of the pharmaceutical composition ranges from approximately a second to approximately twelve hours.

20. The method of any one of claims 1 to 19, wherein the pharmaceutical composition is applied by wiping, brushing, spraying, or use of a sustained-release patch.

21. The method of any one of claims 1 to 20, wherein the pharmaceutical composition is applied once or twice or multiple times a day.

22. The method of any one of claims 1 to 21, wherein the method has a lower drug delivery to anterior segment and a lower systemic drug exposure, as compared to a method administrating the same amount of the muscarinic acetylcholine receptor modulator via eye drop administration into an eye.

23. The method of any one of claims 1 to 22, wherein the ocular drug delivery is used for prevention or treatment of an ocular disorder.

24. The method of claim 23, wherein the ocular disorder is myopia.

25. The method of claim 24, wherein the ocular disorder is child or youth myopia.

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