Use of calcium-sensing receptor antagonists for the treatment of eye diseases

Abstract

This specification discloses therapies for treating eye diseases such as dry eye by targeting calcium-sensing receptors (CaSRs), which are regulators of ocular surface ion transport.

Description

[Technical Field]

[0001] Descriptions concerning government interests This research was supported by grants EY036139, DK126070, EY033859, and EY031372 from the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0002] The tear film covers the cornea and conjunctiva, thereby forming a protective barrier between the external environment and the surface of the eye. 1 Dysfunction in tear production causes dry eye disease (DED), a very common health problem that particularly affects older adults. 2、3 The tear film consists of three layers: the innermost mucin layer, the outermost oil layer, and the large aqueous layer between them. The aqueous layer of the tear film mainly consists of water and electrolytes secreted from the lacrimal glands, cornea, and conjunctiva, and the balance between ion secretion and ion absorption determines the thickness of the tear film. 4 Certain ion channels and ion transporters are involved in ocular surface hydration. Epithelial Na + The channel (ENaC) is Na + While it is the main pathway for fluid absorption, cystic fibrosis membrane conductance regulator (CFTR) is the main pathway for fluid absorption on the ocular surface. - It is also a major pathway for fluid secretion. ENaC and CFTR play important roles in ocular fluid transport and are therefore major targets for DED drug discovery. 5、6 . [Overview of the project]

[0003] In this specification, a method for treating eye diseases caused by reduced ocular surface hydration and / or inflammation is provided by targeting the extracellular calcium-sensing receptor (CaSR), a regulator of ocular surface ion transport. In particular, selective CaSR antagonists such as 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthalenyl)ethyl]amino)-2-hydroxypropoxy]benzonitrile (also known as NPS-2143) are described herein as effective therapies for treating or alleviating the symptoms of DED.

[0004] According to the present disclosure, it has been discovered that CaSR is prominently expressed in the corneas and conjunctivas (including goblet cells) of mice and humans. Furthermore, it has been discovered that CaSR is a major regulator of ocular surface ion transport. The effects of CaSR modulators on ocular surface ion transport were tested in mice by measuring ocular surface potential difference (OSPD) and tear volume. For example, the locally administered CaSR agonist cinacalcet did not affect the baseline OSPD or Na + absorption mediated by the epithelial sodium channel (ENaC). However, cinacalcet inhibited the Cl - secretion and CFTR activity induced by the cAMP agonist forskolin by up to 90% at 30 μM in a concentration-dependent manner. The locally applied CaSR antagonist NPS-2143 elicited a large Cl - secretion current on the ocular surface of mice, and thereafter the forskolin secretory effect was minimal. The effect of NPS-2143 was reversed by the CFTR inhibitor (CFTR inh -172), suggesting the CFTR-dependence of its action. Consistent with these results, a single-dose topical NPS-2143 treatment (0.001% or 30 μM) in mice increased the tear volume in mice by more than 60% for at least 8 hours.

[0005] Therefore, CaSR antagonists increase Cl -And by stimulating body fluid secretion, a novel secretagogue-based therapeutic approach for treating ocular surface diseases (e.g., DED) is provided.

[0006] The present disclosure provides the therapeutic use of a calcium-sensing receptor (CaSR) antagonist for treating ocular surface diseases including one or more dry eye diseases, keratoconjunctivitis, keratitis, or Sjögren's syndrome.

[0007] The present disclosure also provides a topical ophthalmic formulation for treating ocular surface diseases or increasing tear production, the topical ophthalmic formulation comprising a calcium-sensing receptor (CaSR) antagonist and an ophthalmically acceptable excipient.

[0008] The present disclosure also provides a method for treating an ocular surface disease or increasing tear production, the method comprising administering to a subject in need of treatment of an ocular surface disease or increasing tear production a pharmaceutical composition comprising a therapeutically effective amount of a calcium-sensing receptor (CaSR) antagonist and an ophthalmically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] [Figure 1] Mouse ocular surface potential difference (OSPD) measurement setup. [Figure 2] CaSR expression in mouse ocular surface epithelium. Sections of the cornea and conjunctiva were obtained from 12-week-old wild-type BALB / c mice. Anti-CaSR antibody (1:200 dilution) showed immunofluorescent staining in corneal and conjunctival epithelia. Hoechst33258 was used as a nuclear marker. The right photograph shows a negative control without the primary antibody. Scale bar = 25 μm. [Figure 3] CaSR expression in human ocular surface epithelium. Sections of the cornea and conjunctiva were obtained from whole human eyes of patients without a history of visual or ocular diseases. Anti-CaSR antibody (1:200 dilution) showed immunofluorescent staining in corneal and conjunctival epithelia. Hoechst33258 was used as a nuclear marker. The right photograph shows a negative control without the primary antibody. Scale bar = 25 μm. [Figure 4A] The CaSR agonist or activator, cinacalcet, inhibits CFTR-mediated Cl- secretion on the mouse ocular surface. Representative traces of the ocular surface potential difference (OSPD) in mice with control (0.2% DMSO) and various cinacalcet concentrations. [Figure 4B] Aggregation of OSPD data as in A. [Figure 4C] Changes in OSPD (ΔOSPD) induced by 100 μM amiloride in the presence of 0 - 30 μM cinacalcet in the whole solution. [Figure 4D] Changes in OSPD (ΔOSPD) induced by 10 μM forskolin in the presence of 0 - 30 μM cinacalcet in the whole solution. [Figure 4E] Changes in OSPD (ΔOSPD) induced by 10 μM CFTRinh-172 in the presence of 0 - 30 μM cinacalcet in the whole solution. [Figure 4F] Changes in OSPD (ΔOSPD) induced by 100 μM ATP in the presence of 0 - 30 μM cinacalcet in the whole solution. Mean ± standard error, n = 5 - 15 eyes per group, Student's t-test, **p < 0.01, ***p < 0.001, ns: not significant. [Figure 5] Effect of cinacalcet on ATP-induced Cl- on the mouse ocular surface. A. Representative traces of the ocular surface potential difference (OSPD) in mice with and without 30 μM cinacalcet (in the whole solution) in the absence of forskolin and CFTRinh-172. B. Changes in OSPD (ΔOSPD) induced by 100 μM ATP in the experiment of A. Mean ± standard error, n = 8 eyes per group, Student's t-test, **p < 0.01. [Figure 6A] The CaSR antagonist or inhibitor, NPS-2143, stimulates CFTR-mediated Cl- secretion on the mouse ocular surface and increases the tear volume in mice. Representative traces of the ocular surface potential difference (OSPD) showing the effects of 30 μM NPS-2143 followed by 10 μM forskolin and 10 μM CFTRinh-172. [Figure 6B]Summary of OSPD data (left) and changes in OSPD (ΔOSPD, right) as shown in A. Mean ± standard error, n=5 eyes per group. [Figure 6C] Endodontic absorbable paper point tear test (EAPPTT) technique for measuring tear volume in awake, unanesthetized BALB / c mice. The mouse is lightly restrained, and the EAPPTT is placed in the inferior fornix with forceps. [Figure 6D] Tear volume was measured after a single instillation of 10 μL of 0.001% (30 μM) NPS-2143 or an excipient (PBS containing 0.2% DMSO) into BALB / c mice. n=8 eyes per group, Student's t-test, ***p<0.001, compared to the control group at the same time point. [Figure 7] CaSR expression in mouse conjunctival goblet cells. Conjunctival sections were obtained from 12-week-old BALB / c mice. Co-staining of anti-CaSR antibody (1:200 dilution) with anti-cytokeratin 7(K7) antibody (1:200 dilution) showed CaSR immunofluorescence expression in goblet cells. Hoechst33258 was used as a nuclear marker. [Figure 8A] CaSR expression in mouse lacrimal glands. Extraorbital lacrimal gland sections were obtained from 12-week-old mice. Anti-CaSR antibody (1:200 dilution) showed immunofluorescence staining throughout the lacrimal gland. Hoechst33258 was used as a nuclear marker. Scale bar = 25 μm. Staining specificity was confirmed by a negative control without anti-CaSR primary antibody. [Figure 8B] CaSR protein expression in the mouse ocular surface and lacrimal gland. Western blotting was performed on extraorbital lacrimal gland and conjunctival samples from 12-week-old mice. β-actin was used as an internal control in all experiments. Kidney samples from the same animals were used as a positive control to express high levels of CaSR. Primary and secondary antibodies were used at a 1:1000 dilution. This is a representative example from an experiment with n=4. [Figure 9]The CaSR inhibitor NPS-2143 stimulates tear secretion in mice. (A) Standard curves of EAPPTT wet length at different physiological volumes of phosphate-buffered saline (PBS). n=6 per volume, R²=0.9934. (B) Tear volume measurements at indicated time points after a single instillation of 10 μL of 0.001% (30 μM) NPS-2143 or 10 μL of excipient (PBS containing 0.2% DMSO) at zero in BALB / c mice. n=8 eyes per group, Student's t-test, ***p<0.001, compared to control group at the same time point. [Modes for carrying out the invention]

[0010] DED is characterized by reduced ocular surface hydration, which can lead to tissue damage and inflammation. As will be described in more detail below, CaSR targeting offers an effective therapeutic approach to treat DED by increasing fluid secretion and reducing inflammation.

[0011] CaSR is expressed in the corneal and conjunctival epithelium. Extracellular Ca 2+ CaSR receptors are extracellular Ca 2+ It is a G protein-binding receptor that regulates various physiological processes, such as parathyroid hormone secretion, in response to changes in this receptor. 7 CaSR is expressed in various epithelial cells, such as those in the intestines and kidneys, where it regulates ion transport.

[0012] According to this disclosure, CaSR was found to be primarily expressed in epithelial cells of the ocular surface that are in direct contact with the tear film, based on studies in both mice and humans. Immunofluorescence staining of mouse cornea (Figure 2, top) showed marked CaSR expression in the epithelial layer and no expression in the parenchyma. CaSR expression was most prominent in the basal corneal epithelium (most cells were stained positive), and the expression level gradually decreased toward the outermost epithelium.

[0013] It was further discovered that CaSR is strongly expressed in mouse conjunctival epithelium (Figure 2, bottom) but not in the fibrous layer. Furthermore, CaSR was found to be expressed in mouse conjunctival goblet cells (Figure 7).

[0014] CaSR is also known to be primarily expressed in the epithelial layer of the human cornea and conjunctiva, with only minimal expression in the stroma (Figure 3). Similar to mouse corneas, CaSR expression is more pronounced in the basal layer of the human cornea, with most cells expressing CaSR.

[0015] CaSR is expressed in the lacrimal gland. The lacrimal gland is the primary source of tears. According to this disclosure, significant CaSR expression was observed throughout the mouse lacrimal gland after CaSR immunostaining (Figure 8A). Western blotting was used to further confirm CaSR protein expression in the lacrimal gland and ocular surface, and CaSR protein was found to be expressed in both the mouse lacrimal gland and ocular surface. Importantly, CaSR expression was very high in the mouse lacrimal gland (Figure 8B), comparable to that in the positive control tissue of the kidney. These studies further confirm that CaSR is a promising therapeutic target for ocular diseases, including dry eye disease.

[0016] CaSR is the primary regulator of ocular surface ion transport. In this specification, in mouse studies, CaSR plays an important role in ion transport on the ocular surface, specifically, activation of CaSR is associated with Cl - While this leads to a decrease in secretion, inhibition of CaSR leads to CFTR-mediated Cl - It has been demonstrated that it stimulates secretion and increases tear volume.

[0017] 1. The CaSR activator cinacalcet is involved in CFTR-mediated Cl on the mouse eye surface. - Suppressed secretion By using pharmacological activators and inhibitors, OSPD makes it possible to study the activity of various ion channels / ion transporters on the ocular surface. 12In these tests, baseline OSPD mimicked the tear film with high Cl - The solution is established. Changes in OSPD induced by amyloride (an ENaC inhibitor) are due to Na mediated by ENaC. + This indicates transport. Similarly, forskolin (cAMP agonist) and CFTR inh Changes in OSPD induced by -172 (CFTR inhibitor) are due to cAMP activation Cl - Channels, particularly Cl through CFTR - It shows secretion. Finally, Ca 2+ The changes in OSPD induced by the agonist ATP are mediated by CaCC in Cl - It shows secretion.

[0018] Therefore, amiloride (ENaC inhibitor), forskolin (cAMP agonist), CFTR inh -172 (CFTR inhibitor), and ATP (Ca 2+ By continuously perfusing the ocular surface with a solution containing an agonist, it becomes possible to measure the activity of ENaC, CFTR, and CaCC ion channels, respectively (Figure 4A). Cinacalcet treatment did not affect ENaC activity, as suggested by its non-affecting of amyloride-induced depolarization (Figures 4A-4C). However, cinacalcet, at 30 μM, affected forskolin (Figure 4D) and CFTR inh Cinacalcet inhibited CFTR activity in a concentration-dependent manner, as suggested by a maximum 90% reduction in the response at -172 (Figure 4E). In this setting, cinacalcet treatment similarly suppressed CaCC activity, as suggested by a 90% reduction in ATP-induced hyperpolarization (Figure 4F). These results suggest that activation of CaSR (e.g., by cinacalcet) inhibits CFTR-mediated Cl- secretion from the ocular surface but does not affect ENaC-mediated Na+ uptake.

[0019] 2. Cinacalcet is a CaCC-mediated Cl reaction on the surface of the mouse eye. - Suppressed secretion CFTR and CaCC are different intracellular signaling pathways (cAMP and Ca, respectively). 2+ ) is activated by the intracellular cAMP signaling pathway and intracellular Ca 2+ Crosstalk between signaling pathways can lead to confounding effects when activators or inhibitors of these pathways are applied sequentially to the same system. 16 Prior to use, forskolin and CFTR inh In OSPD experiments without treatment with -172, the effect of cinacalcet on CaCC was directly demonstrated. In this setting, cinacalcet was used to treat forskolin and CFTR inh Although the effect was lower than in experiments where ATP was added after -172 (compared to Figure 4F), it still inhibited the ATP response by approximately 50% (Figure 5). These results suggest that the CaSR activator cinacalcet also inhibits CaCC-mediated Cl- secretion on the ocular surface.

[0020] 3. The CaSR antagonist NPS-2143 is CFTR-mediated Cl on the mouse eye surface. - Induces secretion Tear fluid contains 0.4-1.1 mM calcium 2+ This includes plasma ionized Ca 2+ Comparable to 17 Physiological calcium in tears 2+ The concentration is hypothesized to have a sustained antisecretory effect on the ocular surface due to CaSR activation. This hypothesis was confirmed in OSPD experiments using the CaSR antagonist NPS-2143. Perfusion of the ocular surface with 30 μM NPS-2143 resulted in a large hyperpolarization (-13 mV), which was 70% of the maximum forskolin-induced hyperpolarization subsequently applied. Subsequently, the cAMP agonist forskolin showed minimal further secretory effects (Figures 6A and 6B). The responses of NPS-2143 and forskolin were observed in CFTR. inh This is partially reversed by -172, suggesting that the effect is CFTR-dependent. These results suggest that CaSR has a sustained anti-secretory effect on the ocular surface through inhibition of CFTR activity.

[0021] 4. NPS-2143 increases tear volume in mice, and this effect is reversible. The effect of NPS-2143 on tear volume was tested in awake mice using EAPPTT. Topically administered NPS-2143 (0.001% or 30 μM, single 10 μL dose) increased tear volume in mice by 60% as early as 15 minutes after treatment. Importantly, the effect of the single dose of NPS-2143 lasted for at least 6 hours (Figure 6D).

[0022] The effect of NPS-2143 on tear volume is permanent and reversible. In another study using EAPPTT, the accuracy of EAPPTT was confirmed by a standard curve (Figure 9A). Topically administered NPS-2143 (0.001% or 30 μM, single 10 μL dose) increased the tear volume of mice by approximately 60% as early as 15 minutes after treatment (EAPPTT wet length was 2.3 ± 0.2 mm at baseline vs. 3.6 ± 0.2 mm 15 minutes after NPS-2143 administration, mean ± standard error, p < 0.001). The effect of the single dose of NPS-2143 lasted for at least 8 hours compared to the excipient group (EAPPTT wet length was 3.6 ± 0.2 mm 8 hours after NPS-2143 administration). Importantly, the effect of NPS-2143 is reversible, as suggested by the return of tear volume to near baseline values ​​24 hours after treatment (Figure 9B). These results suggest that topical treatment with CaSR antagonists, administered as eye drops two to three times a day, may increase tear volume and enhance the sustained efficacy of NPS-2143.

[0023] Therapeutic use of CaSR antagonists for treating ocular surface diseases According to this disclosure, CaSR is prominently expressed on the ocular surface, including the corneal and conjunctival epithelium of mice and humans. In particular, CaSR is expressed in conjunctival goblet cells, suggesting that CaSR may also be involved in mucin production and / or secretion. CaSR is also expressed in the lacrimal glands, the main source of tears.

[0024] According to this disclosure, CaSR antagonists or inhibitors have a secretion-promoting effect on the ocular surface and possibly also have a secretion effect on the lacrimal gland.

[0025] Considering their secretion-promoting effects, CaSR antagonists may be effective in treating ocular surface diseases, such as dry eye, by increasing tear volume and / or reducing inflammation of dry eye caused by multiple etiologies, including lacrimal gland-targeting diseases such as Sjögren's syndrome.

[0026] Dry eye disease, or DED, refers to a condition in which a patient experiences dryness in one or both eyes. Dry eye disease is characterized by insufficient quality or quantity of tear production. Exemplary symptoms of dry eye disease include irritation, burning, stinging, discharge, foreign body sensation, lacrimation, blurred vision, or a combination of two or more symptoms. Dry eye disease is sometimes instead called dry eye syndrome, keratoconjunctivitis sicca, lacrimal dysfunction syndrome, or dacryokeratoconjunctivitis. Dry eye disease can be caused by multiple etiologies, including medications, aging, rosacea, blepharitis, autoimmune diseases (e.g., Sjögren's syndrome), inflammation (e.g., keratoconjunctivitis and keratitis), diabetes, thyroid disease, vitamin A deficiency, environmental conditions (e.g., dry or windy environments), seasonal allergies, sun exposure, or laser ophthalmic surgery. In this embodiment, dry eye disease may be diagnosed by Schirmer tear film analysis and / or by ocular surface staining patterns of lysamine green, rose bengal, and / or fluorescein dyes.

[0027] "Increasing tear production" or "increasing tear volume" means increasing a patient's tear production compared to a control. The control may be the same patient before treatment, a statistical group of untreated patients, or different untreated patients. In various embodiments, increasing tear production means doubling a patient's tear production compared to (or compared to) a patient's tear production before treatment with the activator described herein. In other embodiments, increasing tear production means tripling or quadrupling a patient's tear production compared to (or compared to) a patient's tear production before treatment with the activator described herein. In embodiments, increasing tear production means increasing a patient's tear production to within the normal range of patient tear production compared to a control or an applicable standard known in the art, including, for example, Schirmer tear tests I (without anesthesia) and II (with anesthesia, measured after local 0.5% propalacaine instillation).

[0028] When used herein, a "CaSR antagonist" or "CaSR inhibitor" effective for the treatment of dry eye according to this disclosure is an IC of 10 micromoles or less relative to CaSR. 50 This includes any compound or drug exhibiting the following characteristics. Examples of CaSR antagonists include, but are not limited to, NPS-2143, NPS-2390, NPSP-795 (or SB-423562), Ronacareret, Encareret (JTT-305), Calcium-Sensing Receptor Antagonist I, Calhex231, Ligstroflavone, SB-423557, and CaSR Antagonist-1. Table 1 below shows the chemical names and structures of these examples. [Table 1-1] [Table 1-2] [Table 1-3]

[0029] Currently approved DED drugs primarily target inflammation and do not correct tear volume or hyperosmolarity, which are major contributing factors to the pathology. 18~21 Several secretion-promoting or antiabsorbable DED therapies targeting ion transport, such as small molecule CFTR activators, are currently in clinical development. 5、6、22~25 Diquafosol is a purinergic P2Y2 receptor agonist that primarily works by increasing CaCC activity on the ocular surface. While diquafosol is approved in Japan, it has not met the primary endpoint in clinical trials, and the FDA has not approved it for DED. Another drawback of diquafosol is that while a long-acting formulation is currently in clinical development, it requires frequent administration (six times a day). 26 .

[0030] In contrast to conventional DED therapy, CaSR antagonists such as NPS-2143 exhibit a sustained secretion-promoting effect in mice for at least 8 hours after a single dose. In addition to the secretion-promoting effect described herein, CaSR antagonists have been shown to have anti-inflammatory effects in other tissues. 27 .

[0031] Accordingly, one embodiment provides a method for treating DED, the method comprising topically administering a pharmaceutical composition comprising a CaSR antagonist and an ophthalmologically acceptable excipient to a subject requiring such treatment.

[0032] A suitable CaSR antagonist may be any calcilytic drug. Calcilytic drugs are usually administered orally, and Ca 2+This causes a rapid increase in plasma levels of PTH, accompanied by an increase in plasma levels of . In a more specific embodiment, the CaSR antagonist is NPS-2143, Ronacareret, or Encareret. In other more specific embodiments, the CaSR antagonist is NPS-2390, NPSP-795 (or SB-423562), calcium-sensing receptor antagonist I, Calhex231, ligstroflavone, SB-423557, or CaSR antagonist-1. Additional examples of calcilic drugs are described, for example, in Nemeth EF et al, Calcif Tissue Int 98:341-358 (2016).

[0033] The CaSR antagonists and pharmaceutical compositions described herein are administered to one or both eyes of the subject. The CaSR antagonists and pharmaceutical compositions can be delivered by any mechanism, such as on the ocular surface (e.g., as eye drops or ointment) or into the eye (e.g., via punctal plugs or subconjunctival injection). In some embodiments, the CaSR antagonists and pharmaceutical compositions are administered topically to the ocular surface. In more specific embodiments, topical administration is topical administration or injection into the conjunctiva of the eye. In embodiments, topical administration is topical administration or injection into the conjunctival sac of the eye. In embodiments, topical administration is topical administration or injection into the conjunctiva and conjunctival sac of the eye. The active agents and compositions described herein can be delivered topically as liquid formulations, for example, as eye drops. In embodiments, the topical liquid formulation is a solution. In embodiments, the topical liquid formulation is an aqueous solution. In embodiments, the topical liquid formulation is a suspension. In embodiments, the topical liquid formulation is an emulsion.

[0034] In other embodiments, the CaSR antagonists and compositions described herein may be delivered into the eye through an implantable device comprising a fluid-eluting contact lens. The implantable device may include a reservoir containing the pharmaceutical compositions described herein and may further include means for enabling the sustained elution of the active agent onto the ocular surface. See, for example, US2020 / 0409177.

[0035] In certain embodiments, ophthalmologically acceptable excipients may include water, NaCl, physiological saline, Ringer's lactate solution, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and pigments. Such preparations may be sterilized and, if desired, mixed with other ophthalmologically acceptable excipients such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants and / or fragrances. [Examples]

[0036] Chemical substances and solutions The composition of the perfusion solution follows the solution used in standardized human nasal and ocular potentiometric protocols. 12、13 Solutions #1-3 (each with high Cl - , amyloride, and low Cl - Solution #1 (high Cl) was prepared in 1-liter batches, the pH was adjusted to 7.4, filtered in a sterile environment before refrigeration, and used within 3 months. - Solution #1 contained 1 L of Ringer's solution (147 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2.4 mM K2HPO4, 0.4 mM KH2PO4, and 1.2 mM MgCl2). 100 μM amyloride was added to solution #1 to prepare solution #2 (amiloride). Solution #3 (low Cl - Solution #1 was identical to Solution #1, except that NaCl was replaced with sodium gluconate. Cinacalcet (CaSR agonist) and NPS-2143 (CaSR antagonist) were purchased from Tocris Bioscience (Minneapolis, MN, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0037] animal BALB / c mice (female and male, 8-12 weeks old) were bred at the UCSF Laboratory Animal Resource Center. The experimental protocol was approved by the UCSF Committee on Animal Experiments. The animal experiments were conducted in compliance with the ARVO statement on the use of animals in ophthalmic and vision research.

[0038] Ocular surface potential difference (OSPD) measurement In mice locally administered cinacalcet (a CaSR activator) and NPS-2143 (a CaSR inhibitor), surface potential difference (OSPD) and tear volume were measured. The mice were anesthetized with isoflurane, and their body temperature was maintained at 37°C via a heating pad. As described above... 12、14、15 Open-circuit transepithelial potential difference (OSPD) was measured in response to continuous perfusion of different isotonic solutions (310 mOsm / kgH2O) onto the ocular surface at 5–10 ml / min. The measurement electrode was in contact with a perfusion catheter carefully positioned directly above the mouse ocular surface using a triaxial micromanipulator, while the reference electrode was grounded via a 23-gauge winged needle agar bridge inserted subcutaneously into the back. Both the measurement and reference electrodes consisted of Ag / AgCl with a 3M KCl agar bridge, and both electrodes were connected to an ISO-Z headstage, a BMA-200 high-impedance amplifier / voltmeter, and a PowerLab analog-to-digital converter (ADInstruments; Colorado Springs, CO, USA) connected to a computer (Figure 1). Each solution was perfused onto the ocular surface for 1–3 minutes using a gravity perfusion system (ALA Scientific; Farmingdale, NY, USA) to obtain stable OSPD readings.

[0039] Immunofluorescence staining CaSR immunostaining was performed on mouse and human corneas and conjunctiva. Sections were obtained from BALB / c mouse eyes and human donors without a history of visual or ocular disease. Mouse and human eyes were embedded in 15 μm frozen sections using a LEICA CM1860 (Leica Biosystems; Deer Park, IL, USA), sectioned, and mounted on Fisherbrand Superfrost Plus microscope slides (Fisher Scientific; Hampton, NH, USA). Sections were stored at -80°C. For immunostaining, sections were brought to room temperature, washed three times with phosphate-buffered saline (PBS) for 5 minutes each, and incubated for 1 hour at room temperature in a humidity chamber with a block containing 50 mM Tris pH 7.4, 100 mM NaCl, 0.1% TX100, 3% normal goat serum, 0.1% BSA, and deionized water. Next, the eye sections were treated overnight at 4°C with anti-CASR antibody [5C10, ADD] (Abcam; Cambridge, UK). The following day, the slides were washed four times for 5 minutes each with a washing solution containing 50 mM Tris pH 7.4, 100 mM NaCl, 0.1% TX100, and deionized water. Secondary antibodies (mouse: Cy3 AffiniPure goat anti-mouse IgG2a, human: Alexa Fluor 488 IgG2a goat anti-mouse (γ2a), Fisher Scientific) were dissolved in a block (1:500) and added at room temperature in a humidified chamber for 2 hours. The slides were washed again four times for 5 minutes each in the washing solution and once for 5 minutes in PBS. The sections were incubated with Hoechst33258 (nuclear marker) for 5 minutes, and then rinsed with deionized water. The slides were mounted using FluoroMount-G (Fisher Scientific) and covered with Corning cover glass 24mm x 50mm (Corning Inc; Corning, NY, USA).

[0040] The control was acquired using a secondary antibody in the absence of a primary antibody, and no immunofluorescence was observed in this setting.

[0041] Measurement of tear volume in mice Using an absorbable paper-point tear test (EAPPTT), 10 μL of PBS containing 30 μM NPS-2143 (or 0.2% DMSO control) was administered as a single drop to both eyes of BALB / c mice, and tear volume was measured at baseline and at 15 minutes, 1 hour, 3 hours, and 6 hours. Unanesthetized mice were loosely fixed while awake, and the EAPPTT (absorbable point, size 30, standard 0.02 taper; Densply Maillefer; Tulsa, OK, USA) was placed in the inferior conjunctival fornix for 20 seconds using forceps. Wetting length was measured using a millimeter-scale ruler under magnification. All mice were kept under ambient conditions (approximately 50% humidity and 21°C) and given free access to food and water.

[0042] statistical analysis Experiments with two groups were analyzed using an unpaired Student's t-test (two-tailed test). For three or more groups, one-way analysis of variance (ANOVA) and post-hoc Newman-Keuls multiple comparison tests were performed using GraphPad Prism (GraphPad Software; Boston, MA, USA). In all analyses, p < 0.05 was considered statistically significant.

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[0044] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or enumerated in the application datasheet are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified as necessary to adopt the concepts of various patents, applications, and publications in order to provide further embodiments.

[0045] In light of the detailed description above, these and other modifications may be made to the embodiments. In general, the terms used in the following claims should not be construed as limiting the claims to the Specified and specific embodiments disclosed herein, but rather as encompassing all possible embodiments, along with the entire scope of equivalents of such claims. Accordingly, the scope of the claims is not limited by this disclosure.

[0046] This application claims priority to U.S. Provisional Application No. 63 / 507,430, filed 9 June 2023, which is incorporated herein by reference in its entirety.

Claims

1. A pharmaceutical composition comprising a calcium-sensing receptor (CaSR) antagonist and an ophthalmologically acceptable excipient for use in the treatment of ocular surface diseases.

2. The pharmaceutical composition described above is the pharmaceutical composition according to claim 1, applied to the target eye.

3. The pharmaceutical composition according to claim 1 or claim 2, wherein the pharmaceutical composition is applied to the target eye every 4 to 12 hours, or preferably every 6 to 8 hours.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the ocular surface disease is one or more dry eye diseases, keratoconjunctivitis, keratitis, or Sjögren's syndrome.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is applied as eye drops, punctal plugs, subconjunctival injections, or via fluid-eluting contact lenses.

6. The CaSR antagonist is 10 μm or less in IC ５０ A pharmaceutical composition according to any one of claims 1 to 5, having a value.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the CaSR antagonist is 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthalenyl)ethyl]amino)-2-hydroxypropoxy]benzonitrile (NPS-2143), Ronacarelet, Encarelet, NPS-2390, NPSP-795 (or SB-423562), calcium-sensing receptor antagonist I, Calhex231, ligstroflavone, SB-423557, or CaSR antagonist-1.

8. A pharmaceutical composition comprising a calcium-sensing receptor (CaSR) antagonist and an ophthalmologically acceptable excipient for use in increasing tear production.

9. The pharmaceutical composition according to claim 8, which is applied to the eye of a target.

10. The pharmaceutical composition according to claim 8 or 9, wherein the pharmaceutical composition is applied to the target eye every 4 to 12 hours, or preferably every 6 to 8 hours.

11. The CaSR antagonist is 10 μm or less in IC ５０ A pharmaceutical composition according to any one of claims 8 to 10, having a value.

12. The pharmaceutical composition according to claim 11, wherein the CaSR antagonist is 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthalenyl)ethyl]amino)-2-hydroxypropoxy]benzonitrile (NPS-2143), Ronacarelet, Encarelet, NPS-2390, NPSP-795 (or SB-423562), calcium-sensing receptor antagonist I, Calhex231, ligstroflavone, SB-423557, or CaSR antagonist-1.

13. A topical ophthalmic preparation for use in the treatment of ocular surface diseases or for increasing tear production, wherein the ophthalmic preparation comprises a calcium-sensing receptor (CaSR) antagonist and an ophthalmologically acceptable excipient.

14. The topical ophthalmic preparation according to claim 13, wherein the ocular surface disease includes dry eye disease, keratoconjunctivitis, keratitis, or Sjögren's syndrome.

15. The CaSR antagonist is 10 μm or less in IC ５０ A topical ophthalmic preparation according to claim 13 or claim 14, having a value, preferably 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthalenyl)ethyl]amino)-2-hydroxypropoxy]benzonitrile (NPS-2143), Ronacarelet, Encarelet, NPS-2390, NPSP-795 (or SB-423562), calcium-sensing receptor antagonist I, Calhex231, ligstroflavone, SB-423557, or CaSR antagonist-1.

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