Use of calcium-sensitive receptor antagonists for treatment of ocular disorders

Abstract

Disclosed herein are therapies for the treatment of ocular disorders, such as dry eye, by targeting calcium-sensitive receptors (CaSR) that are modulators of ocular surface ion transport.

Description

[0001] GOVERNMENT INTEREST STATEMENT

[0002] This work was supported by the National Institutes of Health EY036139, DK126070, EY033859, and EY031372. The government has certain rights in the invention. BACKGROUND

[0003] The tear film covers the cornea and conjunctiva, thereby forming a protective barrier between the external environment and the ocular surface. 1 Impaired tear secretion leads to dry eye disease (DED), a very common health problem that particularly affects the aging population. 2,3

[0004] The tear film is composed of three layers: a mucus layer in the innermost, a lipid layer in the outermost, and a large aqueous layer in the middle. The aqueous layer of the tear film is mainly composed of water and electrolytes secreted by the lacrimal gland, cornea, and conjunctiva, and the balance between ion secretion and absorption determines the tear film height. 4

[0005] Certain ion channels and transporters are involved in ocular surface hydration. The epithelial Na + channel (ENaC) is the main pathway for Na + and fluid absorption, while the cystic fibrosis transmembrane conductance regulator (CFTR) is the main pathway for Cl - and fluid secretion in the ocular surface. Due to their key role in ocular fluid transport, ENaC and CFTR are major targets for DED drug development. 5,6 SUMMARY

[0006] Provided herein are methods of treating ocular conditions caused by reduced ocular surface hydration and / or inflammation by targeting the extracellular calcium-sensing receptor (CaSR), a regulator of ion transport in the ocular surface. In particular, a selective CaSR antagonist such as 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthyl)ethyl]amino)-2-hydroxypropoxy]benzonitrile (also known as NPS-2143) is described herein as an effective therapy for treating or alleviating symptoms of DED.

[0007] According to the present disclosure, it has been discovered that CaSR is significantly expressed in the cornea and conjunctiva, including goblet cells, of mice and humans. It was further discovered that CaSR is a key regulator of ion transport in the ocular surface. The effect of CaSR modulators on ion transport in the ocular surface was tested in mice by measuring ocular surface potential difference (OSPD) and tear volume. For example, the CaSR agonist cinacalcet, administered topically, had no effect on baseline OSDP or Na +Absorption had no effect. However, cinacalcet inhibited the cAMP agonist forskolin-induced CI - secretion and up to 90% of CFTR activity (at 30 µM). Topical application of the CaSR antagonist NPS-2143 caused a large increase in Cl - secretion current, after which forskolin had minimal secretory effect. The effect of NPS-2143 was reversed by the CFTR inhibitor (CFTR inh -172), indicating that its effect was CFTR-dependent. Consistent with these results, a single dose of topical NPS-2143 treatment (0.001% or 30 µM) increased tear volume in mice by >60% for at least eight hours.

[0008] Accordingly, CaSR antagonists provide a novel secretagogue treatment approach for ocular surface disorders (e.g., DED) by stimulating Cl - and fluid secretion from the ocular surface.

[0009] Provided herein are therapeutic uses of a calcium-sensing receptor (CaSR) antagonist for treating ocular surface disorders, including one or more of dry eye disease, keratoconjunctivitis, keratitis, or Sjogren's syndrome (SS).

[0010] Also provided are topical ophthalmic formulations for use in treating ocular surface disorders or increasing tear production, the topical ophthalmic formulations comprising a calcium-sensing receptor (CaSR) antagonist and an ophthalmically acceptable excipient.

[0011] Also provided are methods for treating ocular surface disorders or increasing tear production, the methods comprising: administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a calcium-sensing receptor (CaSR) antagonist and an ophthalmically acceptable excipient. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 . Mouse ocular surface potential difference (OSPD) measurement setup.

[0013] Figure 2 . CaSR expression in mouse ocular surface epithelium. Corneal and conjunctival sections were obtained from 12-week-old wild-type BALB / c mice. Anti-CaSR antibody (1 :200 dilution) showed immunofluorescent staining in the corneal and conjunctival epithelium. Hoechst 33258 was used as a nuclear marker. The right panel shows negative control without primary antibody. Scale bar = 25 µm.

[0014] Figure 3CaSR expression in human ocular surface epithelium. Corneal and conjunctival sections were obtained from eye globe samples of patients with no visual or ocular disease history. Anti-CaSR antibody (1 :200 dilution) showed immunofluorescence staining in corneal and conjunctival epithelium. Hoechst 33258 was used as a nuclear marker. The right panel shows negative control without primary antibody. Scale bar = 25 μm.

[0015] Figure 4. CaSR agonist or activator cinacalcet inhibits CFTR-mediated Cl - secretion in the mouse ocular surface. A. Representative ocular surface potential difference (OSPD) traces in mice in the presence of control (0.2% DMSO) and different concentrations of cinacalcet. B. Summary of the OSDP data as described in A. C-F. Changes in OSDP (Δ OSDP) induced by 100 μΜ amiloride (C), 10 μΜ furosemide (D), 10 μΜ CFTR inh -172 (E), and 100 μΜ ATP (F) in the presence of 0-30 μΜ cinacalcet in all solutions. Mean ± S.E.M., n = 5-15 eyes / group, Student's t-test. t p < 0.01, p < 0.001, ns: not significant.

[0016] Figure 5. Effect of cinacalcet on ATP-induced Cl - secretion in the mouse ocular surface. A. Representative ocular surface potential difference (OSPD) traces in mice using and not using 30 μΜ cinacalcet (in all solutions) in the absence of furosemide and CFTR inh -172. B. Changes in OSDP (Δ OSDP) induced by 100 μΜ ATP in the experiments of A. Mean ± S.E.M., n = 8 eyes / group, Student's t-test. t p < 0.01.

[0017] Figure 6. CaSR antagonist or inhibitor NPS-2143 stimulates CFTR-mediated Cl - ocular surface secretion in mice and increases tear volume. A. Representative ocular surface potential difference (OSPD) traces demonstrating the effect of 30 μΜ NPS-2143, followed by 10 μΜ furosemide and 10 μΜ CFTR inh ​​-172. B. Summary of OSPD data consistent with A (left) and OSPD variation (ΔOSPD, right). Mean ± SEM, n = 5 eyes / group. C. EAPPTT technique for measuring tear volume in awake, unanesthetized BALB / c mice. The mice were gently held still, and the EAPPTT was placed in the lower fornix with forceps. D. Tear volume measurement in BALB / c mice after a single 10 μL drop of 0.001% (30 μM) NPS-2143 eye drops or a medium (PBS containing 0.2% DMSO). n = 8 eyes / group, Student's t-test. p<0.001, compared with the control group at the same time point.

[0018] Figure 7 CaSR expression in goblet cells of the mouse conjunctiva. Conjunctival sections were obtained from 12-week-old BALB / c mice. Immunofluorescence expression of CaSR in goblet cells was demonstrated by co-staining with anti-CaSR antibody (1:200 dilution) and anti-cytokeratin 7 (K7) antibody (1:200 dilution). Hoechst 33258 was used as a nuclear marker.

[0019] Figure 8A CaSR expression in mouse lacrimal glands. Extraorbital lacrimal gland sections were obtained from 12-week-old mice. Immunofluorescence staining was performed throughout the lacrimal gland using anti-CaSR antibody (1:200 dilution). Hoechst 33258 was used as a nuclear marker. Scale bar = 25 µm. A negative control without anti-CaSR primary antibody confirmed the staining specificity.

[0020] Figure 8B CaSR protein expression in the ocular surface and lacrimal glands of mice. Western blot experiments were 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 positive controls for high levels of CaSR expression. Primary and secondary antibodies were used at a dilution of 1:1000. A representative n=4 experiments were performed.

[0021] Figure 9 CaSR inhibitor NPS-2143 stimulates tear secretion in mice. (A) Standard curves of EAPPTT wetting length in different physiological volumes of phosphate-buffered saline (PBS). n=6 / physiological volume, R2=0.9934. (B) Tear volume was measured in BALB / c mice at specific time points after a single instillation of 10 µL of 0.001% (30 µM) NPS-2143 or 10 µL of eye drop medium (PBS containing 0.2% DMSO) at time zero. n=8 eyes / group, Student's t-test. p<0.001, compared with the control group at the same time point. Detailed Implementation

[0022] DED is characterized by reduced ocular surface hydration, which can lead to tissue damage and inflammation. As described in more detail below, targeting CaSR provides an effective treatment for DED by increasing fluid secretion and reducing inflammation.

[0023] CaSR expression in corneal and conjunctival epithelium

[0024] Extracellular Ca 2+ The sensitive receptor (CaSR) is a G protein-coupled receptor that regulates various physiological processes, such as parathyroid hormone secretion, in response to extracellular Ca2+. 2+ The changes. 7 CaSR is expressed in various epithelial cells such as the intestine and kidney, where it plays a role in regulating ion transport.

[0025] According to this disclosure, based on mouse and human studies, CaSR has been found to be primarily expressed in the ocular surface epithelial cells that are in direct contact with the tear film. Immunofluorescence staining in mouse cornea ( Figure 2 The top (top) shows significant CaSR expression in the epithelial layer, while it is absent in the stroma. CaSR expression is most pronounced in the basal corneal epithelium (most cells are positively stained), and the expression level gradually decreases towards the apex of the epithelium.

[0026] Further investigation has revealed that CaSR is strongly expressed in mouse conjunctival epithelium. Figure 2 (bottom), but not in the fibrous layer. Additionally, CaSR was found to be expressed in mouse conjunctival goblet cells (bottom). Figure 7 ).

[0027] It was also found that CaSR was mainly expressed in the epithelial layer of the human cornea and conjunctiva, with the least expression in the stroma. Figure 3 Similar to mouse cornea, CaSR expression is more pronounced in the basal layer of human cornea, where it is expressed by most cells.

[0028] CaSR expression in lacrimal glands

[0029] The lacrimal gland is the main source of tears. According to this disclosure, significant CaSR expression has been found throughout the entire lacrimal gland after CaSR immunostaining in the mouse lacrimal gland. Figure 8A To further confirm the expression of CaSR protein in the lacrimal gland and ocular surface, Western blotting was used, revealing that CaSR protein is expressed in both the mouse lacrimal gland and ocular surface. Importantly, CaSR expression is very high in the mouse lacrimal gland. Figure 8BThe levels of CaSR were comparable to those in the kidneys (positive control tissue). These studies further validate that CaSR is a therapeutic target for eye diseases, including dry eye.

[0030] CaSR is a key regulator of ion transport on the ocular surface.

[0031] This study demonstrates in mice that CaSR plays a crucial role in ion transport on the ocular surface, specifically that CaSR activation leads to a reduction in Cl-. - Secretion, while CaSR inhibits CFTR-mediated Cl- secretion. - It secretes and increases the amount of tears.

[0032] 1. CaSR activator cinacalcet inhibits CFTR-mediated Cl- on the mouse ocular surface - secretion

[0033] By using drug activators and inhibitors, OSPD allows for the study of the activity of various ion channels / transporters on the ocular surface. 12 In these studies, high Cl- levels were used to simulate tear films. - A baseline OSPD was established using a solution. Amiloride (an ENaC inhibitor)-induced OSPD changes indicated Na+ + Transported via ENaC. Similarly, foscorine (a cAMP agonist) and CFTR... inh -172 (CFTR inhibitor)-induced OSPD changes indicate that Cl - Cl activated by cAMP - Channels, especially those secreted by CFTR. Finally, Ca... 2+ ATP-induced OSPD changes indicate that Cl - Secretion is mediated by CaCCs.

[0034] Using products containing amiloride (ENaC inhibitor), foscorlin (cAMP agonist), and CFTR inh -172 (CFTR inhibitor) and ATP (Ca 2+ The agonist solution was sequentially infused into the ocular surface, thus allowing for the separate determination of the activity of ENaC, CFTR, and CaCC ion channels. Figure 4A Cinacalcet treatment has no effect on ENaC activity, as indicated by its lack of effect on amiloride-induced depolarization. Figure 4A -C). However, as with foscorine ( Figure 4D ) and CFTR inh -172 ( Figure 4EThe reduction of up to 90% in the response at 30 μM indicates that cinacalcet concentration-dependent inhibition of CFTR activity. In this case, the 90% reduction in ATP-induced hyperpolarization suggests that cinacalcet treatment similarly inhibits CaCC activity. Figure 4F The results showed that CaSR activation (e.g., with cinalcalcet activation) inhibited CFTR-mediated Cl-. - Ocular surface secretion, but resistant to ENaC-mediated Na+. + Absorption is ineffective.

[0035] 2. Sinacalcet inhibits CaCC-mediated Cl- - mouse ocular surface secretion

[0036] Although CFTR and CaCC are detected by different intracellular signaling pathways (cAMP and Ca, respectively), 2+ ) activation, but intracellular cAMP and Ca 2+ Crosstalk between pathways can lead to confounding effects when activators or inhibitors of these pathways are applied sequentially in the same system. 16 Without prior use of foscorine and CFTR inh OSPD experiments performed under -172 treatment directly demonstrated the effect of cinacalcet on CaCC. Under these conditions, cinacalcet still inhibited the ATP response by approximately 50% (Figure 5), although its effect was less than that achieved with foscorine and CFTR. inh The experiment after adding ATP at -172 (and) Figure 4F (Comparison). These results indicate that the CaSR activator cinacalcet also inhibits CaCC-mediated ocular surface Cl-. - secretion.

[0037] 3. CaSR antagonist NPS-2143 induces CFTR-mediated Cl- on the mouse ocular surface - secretion

[0038] Tears contain 0.4-1.1 mM Ca 2+ This is related to the ionized Ca in the blood plasma 2+ quite. 17 It is hypothesized that, due to the activation of CaSR, physiological Ca in tears... 2+ Concentrations produce a sustained anti-secretion effect on the ocular surface. 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 large hyperpolarization (-13 mV), which was 70% of the maximum hyperpolarization induced by subsequently applied forscorin, after which the cAMP agonist forscorin exhibited minimal further secretion. Figure 6A and 6BNPS-2143 and foscorine reacted with CFTR inh The partial reversal of -172 indicates that its action is CFTR-dependent. These results suggest that CaSR has a sustained antisecretory effect on the ocular surface by inhibiting CFTR activity.

[0039] 4. NPS-2143 increases tear production in mice, and this effect is reversible.

[0040] The effect of NPS-2143 on tear volume was tested in awake mice using EAPPTT. Topical application of NPS-2143 (0.001% or 30 µM, single 10 µL dose) resulted in a 60% increase in tear volume in mice as early as 15 minutes after treatment. Importantly, the effect of a single dose of NPS-2143 lasted for at least 6 hours. Figure 6D ).

[0041] The effect of NPS-2143 on tear volume is persistent and reversible. In another EAPPTT test, the accuracy of EAPPTT was confirmed by a standard curve. Figure 9 A). Topical application of NPS-2143 (0.001% or 30 μM, single 10 μL dose) resulted in an approximately 60% increase in tear volume in mice as early as 15 minutes after treatment (EAPPTT wetting length at baseline: 2.3 ± 0.2 mm vs. 3.6 ± 0.2 mm at 15 minutes after NPS-2143 application, mean ± SEM, p < 0.001). The effect of a single dose of NPS-2143 lasted for at least 8 hours (EAPPTT wetting length 3.6 ± 0.2 mm at 8 hours after NPS-2143 application). Importantly, the effect of NPS-2143 was reversible, as indicated by the recovery of tear volume to near baseline values ​​24 hours after treatment. Figure 9 B). These results suggest that topical treatment with a CaSR antagonist can increase tear volume, and that NPS-2143 has the potential for sustained efficacy when applied as eye drops 2–3 times daily.

[0042] CaSR antagonists are used for the treatment of ocular surface diseases.

[0043] According to this disclosure, CaSR is significantly 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 play a role in mucin production and / or secretion. CaSR is also expressed in the lacrimal glands (the primary source of tears).

[0044] According to this disclosure, CaSR antagonists or inhibitors have a secretory-promoting effect on the ocular surface and may also have a secretory-promoting effect on the lacrimal gland.

[0045] Given their secretory-promoting effects, CaSR antagonists are effective in treating ocular surface conditions, such as dry eye disease, by increasing tear production and / or reducing inflammation. Dry eye diseases caused by a variety of factors include those affecting the lacrimal glands, such as Sjögren's syndrome (SS).

[0046] "Dry eye disease," or DED, refers to a condition in which one or both eyes experience dryness. Dry eye disease is characterized by insufficient tear production or poor tear quality. Exemplary symptoms of dry eye disease include irritation, burning, stinging, discharge, foreign body sensation, tearing, blurred vision, or a combination of two or more symptoms. Dry eye disease may also be referred to as dry eye syndrome, keratoconjunctivitis sicca, dysfunctional tear syndrome, or lacrimal keratoconjunctivitis. Dry eye disease can be caused by a variety of factors, including medications, advanced age, rosacea, blepharitis, autoimmune diseases (e.g., Sjögren's syndrome (SS)), 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 eye surgery. In implementation, dry eye disease can be diagnosed by the Schirmer tear test and / or ocular surface staining patterns using erythromycin green, cinnabar, and / or fluorescein dyes.

[0047] "Increased tear production" or "increased tear volume" refers to an increase in a patient's tear production relative 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, increased tear production means doubling a patient's tear production compared to before treatment with the active agent described herein (or compared to another control). In other embodiments, increased tear production means a three-fold or four-fold increase in tear production compared to a patient's tear production before treatment with the active agent described herein (or compared to another control). In embodiments, increased tear production means increasing a patient's tear production to within the normal range for tear production relative to a control or applicable standards known in the art (including, for example, the Schirmer tear test I (unanesthesia) and II (anesthesia, measured after topical instillation of 0.5% promecaine)).

[0048] As used herein, a “CaSR antagonist” or “CaSR inhibitor” effective in treating dry eye disease according to this disclosure includes any substance that exhibits an IC50 response of 10 μmol or less to CaSR. 50Compounds or drugs of interest. Examples of CaSR antagonists, but not limited to, include NPS-2143, NPS-2390, NPSP-795 (or SB-423562), ronacaleret, encaleret (JTT-305), calcium receptor antagonist I, Calhex 231, ligustroflavone, SB-423557, and CaSR antagonist-1. Table 1 below shows the chemical names and structures of these examples.

[0049] Table 1

[0050] Currently approved DED drugs primarily target inflammation and cannot correct tear volume or hyperosmolarity, which are major drivers of pathology. 18-21 Several DED therapies targeting ion transport, such as secretion-promoting or anti-absorption agents, are currently under clinical development, including small molecule CFTR activators. 5,6,22-25 Dequafosol is a purinergic P2Y2 receptor agonist that primarily works by increasing CaCC activity on the ocular surface. Although approved in Japan, dequafosol failed to meet its primary endpoint in clinical trials and has not been approved by the FDA for diabetic retinopathy (DED). Another drawback of dequafosol is that it requires frequent administration (six times a day), although long-acting formulations are currently in clinical development. 26

[0051] In contrast to conventional DED therapy, CaSR antagonists such as NPS-2143 exhibited sustained secretagogue effects in mice for at least 8 hours following a single dose. In addition to the secretagogue effects described herein, CaSR antagonists have also been shown to have anti-inflammatory effects in other tissues. 27

[0052] Therefore, one embodiment provides a method for treating DED, comprising topical application of a pharmaceutical composition comprising a CaSR antagonist and an ophthalmologically acceptable excipient to a subject in need.

[0053] Suitable CaSR antagonists can be any calcium dissociation drug. Calcium dissociation drugs are usually administered orally and cause a rapid increase in plasma PTH levels, accompanied by an increase in plasma Ca2+. 2+The level increases. In a more specific embodiment, the CaSR antagonist is NPS-2143, ronacaleretide, or encalitide. In other more specific embodiments, the CaSR antagonist is NPS-2390, NPSP-795 (or SB-423562), calcium-sensitive receptor antagonist I, Calhex 231, privetin, SB-423557, or CaSR antagonist-1. Further examples of calcium dissociation drugs are described, for example, by Nemeth EF et al. Calcif Tissue Int 98:341–358 (2016).

[0054] The CaSR antagonist and pharmaceutical composition described herein will be administered to one or both eyes of a subject. The CaSR antagonist and pharmaceutical composition may be delivered via any mechanism, including delivery to 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 antagonist and pharmaceutical composition are applied topically to the ocular surface. In more specific embodiments, topical application is application or injection to the conjunctiva of the eye. In embodiments, topical application is application or injection to the conjunctival sac of the eye. In embodiments, topical application is application to both the conjunctiva and the conjunctival sac of the eye. The active agents and compositions described herein may 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.

[0055] In other embodiments, the CaSR antagonists and compositions described herein can be delivered via an implantable device in the eye, including a liquid-release contact lens. The implantable device includes a reservoir containing the pharmaceutical composition described herein and may further include means that allow the active agent to be released continuously onto the ocular surface. See, for example, U.S. Patent 2020 / 0409177.

[0056] In some implementations, ophthalmologically acceptable excipients may include water, NaCl, physiological saline solutions, lactated Ringer's 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, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments. Such preparations can be sterilized and, if desired, can be mixed with other pharmaceutically acceptable excipients such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, and / or aromatic substances.

[0057] Example

[0058] Chemicals and solutions

[0059] The composition of the perfusion solution follows the solutions used in the standardized human nose and eye potential difference protocol. 12,13 Solution #1-3 (high Cl) - Amiloride and low-chlorine - Prepared in 1-liter batches, pH equilibrated to 7.4, filtered under sterile conditions before refrigeration, and used within 3 months. Solution #1 (high Cl) - The solution contains 1 L of Ringer's injection (containing 147 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2.4 mM K2HPO4, 0.4 mM KH2PO4, and 1.2 mM MgCl2). 100 µM amiloride is added to solution #1 to prepare solution #2 (amiloride). Solution #3 (low Cl...) - The solution was the same as 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).

[0060] animal

[0061] BALB / c mice (female and male, 8–12 weeks old) were housed at the UCSF Laboratory Animal Resource Center. The experimental protocol was approved by the UCSF Institutional Animal Care and Use Committee. Animal experiments were conducted in accordance with ARVO's statement on the use of animals in ophthalmological and vision studies.

[0062] Measurement of ocular surface potential difference (OSPD)

[0063] Ocular surface potential difference (OSPD) and tear volume were measured in mice treated with topical cinacalcet (a CaSR activator) and NPS-2143 (a CaSR inhibitor). Mice were anesthetized with isoflurane and their body temperature was maintained at 37°C using a heating pad. As previously described, 12,14,15Open-circuit transepithelial potential difference was measured in response to continuous perfusion of the ocular surface with different isotonic (310 mOsm / kg H2O) solutions at a rate of 5–10 ml / min. The measuring electrode was in contact with the perfusion catheter, which was carefully positioned directly above the mouse ocular surface using a triaxial micromanipulator. The reference electrode was grounded via a 23-gauge butterfly needle agar bridge inserted subcutaneously into the back of the eye. Both the measuring and reference electrodes consisted of Ag / AgCl and 3 M KCl agar bridges, and both electrodes were connected to an ISO-Z probe, 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 infused onto the ocular surface for 1–3 minutes using a gravity perfusion system (ALA Scientific; Farmingdale, NY, USA) until a stable OSPD reading was obtained.

[0064] Immunofluorescence staining

[0065] CaSR immunostaining was performed on the cornea and conjunctiva of mice and humans. Sections were obtained from BALB / c mouse eyes and human donors with no history of visual or ocular disease. Mouse and human eyes were embedded and cut into 15 µm frozen sections using a LEICA CM1860 (Leica Biosystems; Deer Park, IL, USA) and mounted on Fisherbrand Superfrost Plus microscope slides (Fisher Scientific; Hampton, NH, USA). Sections were stored at -80°C. For immunostaining, sections were placed at room temperature, washed three times with phosphate-buffered saline (PBS) for 5 minutes each time, and incubated for 1 hour at room temperature in a humidified chamber with blocking buffer containing 50 mM Tris pH 7.4, 100 mM NaCl, 0.1% TX100, 3% normal goat serum, 0.1% BSA, and deionized water. The eye sections were then treated overnight at 4°C with anti-CASR antibody [5C10, ADD] (Abcam; Cambridge, UK). The next day, the slides were washed four times for 5 minutes each time in 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) Dissolve the slide in blocking solution (1:500) and place in a humidity chamber at room temperature for 2 hours. Wash the slide four times for 5 minutes each in washing solution, and once for 5 minutes in PBS. Incubate the slide with Hoechst 33258 (nucleus marker) for 5 minutes, then rinse with deionized water. Fix the slide with Fluoromount-G (Fisher Scientific) and cover with Corning coverslips (24 mm x 50 mm, Corning Inc; Corning, NY, USA).

[0066] A control was obtained using a secondary antibody in the absence of a primary antibody, which confirmed the absence of immunofluorescence in this case.

[0067] Mouse tear volume measurement

[0068] The EAPPTT test was used to measure tear volume in BALB / c mice at baseline and bilaterally after administration of a single drop of 10 μL PBS containing 30 μM NPS-2143 (or 0.2% DMSO control). Awake, unanesthetized mice were gently restrained, and the EAPPTT (absorption point, size 30, standard 0.02 taper; Densply Maillefer; Tulsa, OK, USA) was placed in the inferior conjunctival fornix for 20 seconds using forceps. The wetting length was measured using a millimeter ruler observed under a magnifying glass. All mice were held under ambient conditions (approximately 50% humidity and 21°C) and provided with unrestricted food and water.

[0069] Statistical analysis

[0070] Two-tailed unpaired Student's t-tests were used to analyze experiments involving two groups; for three or more groups, analyses were performed using GraphPadPrism (GraphPad software; Boston, MA, USA) via one-way ANOVA and post-hoc Newman-Keuls multiple comparison tests. In all analyses, p < 0.05 was considered statistically significant.

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[0103] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the application data sheets are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to incorporate the concepts of various patents, applications, and publications to provide other embodiments.

[0104] Based on the detailed description above, these and other changes can be made to the embodiments. Generally, the terminology used in the appended claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of their equivalents. Therefore, the claims are not limited by this disclosure.

[0105] This application claims priority to U.S. Provisional Application No. 63 / 507,430, filed June 9, 2023, the entire contents of which are incorporated herein by reference.

Claims

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

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is applied to the eye of the subject.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the pharmaceutical composition is applied to the eyes of the subject every 4-12 hours, or preferably every 6-8 hours.

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

5. The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical composition is administered as an eye drop, a punctal plug, a subconjunctival injection, or via a liquid-release contact lens.

6. The pharmaceutical composition of any one of claims 1-5, wherein the CaSR antagonist has an IC50 of 10 µM or less. 50 value.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the CaSR antagonist is 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthyl)ethyl]amino)-2-hydroxypropoxy]benzyl nitrile (NPS-2143), ronacaleretide, encalitone NPS-2390, NPSP-795 (or SB-423562), calcium-sensitive receptor antagonist I, Calhex 231, ligustrazine, SB-423557, or CaSR antagonist-1.

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

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is applied to the eye of the subject.

10. The pharmaceutical composition of claim 8 or claim 9, wherein the pharmaceutical composition is applied to the eyes of the subject every 4-12 hours, or preferably every 6-8 hours.

11. The pharmaceutical composition of any one of claims 8-10, wherein the CaSR antagonist has an IC50 of 10 µM or less. 50 value.

12. The pharmaceutical composition of claim 11, wherein the CaSR antagonist is 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthyl)ethyl]amino)-2-hydroxypropoxy]benzyl nitrile (NPS-2143), ronacaleretide, encalitone NPS-2390, NPSP-795 (or SB-423562), calcium receptor antagonist I, Calhex 231, ligustrazine, SB-423557, or CaSR antagonist-1.

13. A topical ophthalmic preparation for use in treating ocular surface conditions or increasing tear production, said topical ophthalmic preparation comprising a calcium-sensitive receptor (CaSR) antagonist and ophthalmologically acceptable excipients.

14. The topical ophthalmic preparation of claim 13, wherein the ocular surface condition includes dry eye, keratoconjunctivitis, keratitis, or Sjögren's syndrome (SS).

15. The topical ophthalmic formulation of claim 13 or claim 14, wherein the CaSR antagonist has an IC50 concentration of 10 μM or less. 50 The value, and preferably 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthyl)ethyl]amino)-2-hydroxypropoxy]benzyl nitrile (NPS-2143), ronacaleretide, encalitide NPS-2390, NPSP-795 (or SB-423562), calcium-sensitive receptor antagonist I, Calhex 231, privetin, SB-423557 or CaSR antagonist-1.

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