ASSAY FOR RAPID EVALUATION OF CHOROIDAL MAST CELL DEGRANULATION
20220397569 · 2022-12-15
Inventors
Cpc classification
International classification
Abstract
The present invention relates to the field of ophthalmology. More specifically, the present invention provides compositions and methods useful for screening for drugs to treat age-related macular degeneration (AMD) including geographic atrophy (GA). In one embodiment, a method comprises the steps of (a) administering a drug to a mammal, wherein the mammal comprises a rat or a mouse; (b) enucleating the eyes of the mammal; (c) removing the anterior eye and excising the retina from the eye, wherein the eye comprises an eyecup that comprises choroidal mast cells (MCs); and (d) measuring mast cell degranulation. In an alternative embodiment, a method of the present invention can comprise the steps of (a) contacting an eyecup of a mammal with a drug, wherein the eyecup comprises choroidal mast cells; and (b) measuring MC degranulation.
Claims
1. A method comprising the steps of: (a) administering a drug to a mammal, wherein the mammal comprises a rat or a mouse; (b) enucleating the eyes of the mammal; (c) removing the anterior eye and excising the retina from the eye, wherein the eye comprises an eyecup that comprises choroidal mast cells (MCs); and (d) measuring mast cell degranulation.
2. The method of claim 1, wherein administering step (a) further comprises administering an agent that promotes MC degranulation.
3. The method of claim 2, wherein the agent comprises compound 48/80 or calcium ionophore.
4. The method of claim 1, wherein step (d) comprises staining for non-specific esterase (NSE) activity in mast cells present in the choroid of the eyecup.
5. The method of claim 4, further comprising counting non-degranulated and granulated cells in the whole choroid, wherein degranulated cells exhibit irregular shape or extracellular granules.
6. The method of claim 1, further comprising staining macrophages and measuring macrophage cell volume and sphericity.
7. The method of claim 1, wherein macrophages are stained with anti-Iba1 antibody.
8. The method of claim 1, further comprising staining for tryptase with an anti-MC tryptase antibody.
9. A method comprising the steps of: (a) contacting an eyecup of a mammal with a drug, wherein the eyecup comprises choroidal mast cells; and (b) measuring MC degranulation
10. The method of claim 9, wherein the eyecup has been separated from the anterior eye and retina.
11. The method of claim 9, wherein contacting step (a) further comprises contacting the eyecup with an agent that promotes MC degranulation.
12. The method of claim 11, wherein the agent comprises compound 48/80 or calcium ionophore.
13. The method of claim 9, wherein step (b) comprises staining for NSE activity in mast cells present in the choroid of the eyecup.
14. The method of claim 13, further comprising counting non-degranulated and granulated cells in the whole choroid, wherein degranulated cells exhibit irregular shape or extracellular granules.
15. The method of claim 9, wherein the method further comprises staining macrophages and measuring macrophage cell volume and sphericity.
16. The method of claim 15, wherein macrophages are stained with anti-Iba1 antibody.
17. The method of claim 9, further comprising staining for tryptase with an anti-MC tryptase antibody.
18. The method of claim 9, wherein the time between steps (a) and (b) comprises at least 90 minutes.
19. The method of claim 9, wherein the time between steps (a) and (b) comprises about 180 minutes.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0024] It is understood that the present invention is not limited to the particular methods and components, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a “protein” is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth.
[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
[0026] All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention.
[0027] Recently, the present inventors observed an increase in number and percentage of degranulated choroidal MCs in all types of AMD subjects..sup.13 The possible reason for this is the proinflammatory milieu of AMD choroid, which includes elevated complement factor C3a, C5a, C-reactive protein (CRP), and advanced glycation end products (AGEs), all of which can stimulate MC degranulation..sup.14-16 Human choroidal MCs release tryptase and histamine when they degranulate,.sup.17 and tryptase is released specifically from MCs in the first wave of granules during degranulation..sup.18 Tryptase degrades collagens and activates MMPs, which degrade stroma and basement membranes. In GA, MC-derived tryptase was localized to Bruch's membrane, vascular basement membranes and throughout the choroidal stroma near degranulated MCs..sup.17 These observations imply MC involvement in GA etiology, however, there is still a void in the understanding of how MC degranulation contributes to the progression of GA.
[0028] The purpose of the current study was to determine if chronic choroidal MC degranulation results in changes that were phenotypic characteristics of GA. Therefore, RPE atrophy as well as reduced retinal function, and retinal and choroidal thinning were assessed after inducing MC degranulation in a rat model. To evaluate if MCs could be a therapeutic target in GA, ketotifen fumarate, a generic MC stabilizer, was evaluated for prevention of MC degranulation in in vitro and ex vivo assays for MC degranulation, and in the present inventors' in vivo long-term rat model of GA. In addition, a MC tryptase inhibitor was also evaluated in the rat model. The present inventors' results suggest for the first time, choroidal MC involvement in the development of GA and MCs as a potential therapeutic target for GA.
[0029] Accordingly, in one aspect, the present invention provides compositions and methods useful for screening drugs that stabilize MCs and thus, prevent degranulation. In one embodiment, a method comprises the steps of (a) administering a drug to a mammal, wherein the mammal comprises a rat or a mouse; (b) enucleating the eyes of the mammal; (c) removing the anterior eye and excising the retina from the eye, wherein the eye comprises an eyecup that comprises choroidal mast cells (MCs); and (d) measuring mast cell degranulation.
[0030] In another embodiment, administering step (a) further comprises administering an agent that promotes MC degranulation. In a specific embodiment, the agent comprises compound 48/80.
[0031] In a specific embodiment, step (d) comprises staining for non-specific esterase (NSE) activity in mast cells present in the choroid of the eyecup. In a more specific embodiment, the method further comprises counting non-degranulated and granulated cells in the whole choroid, wherein degranulated cells exhibit irregular shape or extracellular granules
[0032] In another embodiment, the method further comprises staining macrophages and measuring macrophage cell volume and sphericity. In a specific embodiment, macrophages are stained with anti-Iba1 antibody.
[0033] In yet another embodiment, the method further comprises staining for tryptase with an anti-MC tryptase antibody.
[0034] In particular embodiments, the drug is administered to the mammal at least twice over the course of 1-7 days.
[0035] A method of the present invention can comprise the steps of (a) contacting an eyecup of a mammal with a drug, wherein the eyecup comprises choroidal mast cells; and (b) measuring MC degranulation. In particular embodiments, the eyecup has been separated from the anterior eye and retina.
[0036] In another embodiment, contacting step (a) further comprises contacting the eyecup with an agent that promotes MC degranulation. In a specific embodiment, the agent comprises compound 48/80.
[0037] In a specific embodiment, step (b) comprises staining for NSE activity in mast cells present in the choroid of the eyecup. In a more specific embodiment, the method further comprises counting non-degranulated and granulated cells in the whole choroid, wherein degranulated cells exhibit irregular shape or extracellular granules.
[0038] In another embodiment, the method further comprises staining macrophages and measuring macrophage cell volume and sphericity. In a specific embodiment, macrophages are stained with anti-Iba1 antibody.
[0039] In another embodiment, the method further comprises staining for tryptase with an anti-MC tryptase antibody.
[0040] In particular embodiments, the time between steps (a) and (b) comprises at least 90 minutes. In a more specific embodiment, the time between steps (a) and (b) comprises about 180 minutes.
[0041] The methods of the present invention can include administering mast cell stabilizers as a control. Examples of such compounds include, but are not limited to, β2-adrenergic agonists, cromoglicic acid, ketotifen, methylxanthines, olopatadine, rupatidine, mepolizumab, omalizumab, pemirolast, quercetin, nedocromil, azelastine, tranilast, palmitoylethanolamide, and vitamin D.
[0042] Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.
Examples
[0043] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
[0044] The experiments described below sought to determine if mast cells (MCs) contribute to geographic atrophy (GA), an advanced form of age-related macular degeneration. The cause of GA is elusive and there is currently no therapy for this blinding disorder. Mast cells (MCs) are the initial responders of innate immunity and are involved in many diseases. This study demonstrates that choroidal MC degranulation and MC-derived tryptase could be involved in both human GA and a rat model for GA. Continuous activation of MCs in a slow release fashion in the rat induced characteristic phenotypes of human GA: retinal pigment epithelium degeneration followed by retinal and choroidal thinning. Pharmacological intervention targeting MC degranulation with ketotifen fumarate or inhibition of MC-derived tryptase with APC 366 reduced MC degranulation and prevented all of GA-like phenotypes in the rat model. These results suggest choroidal MC involvement in GA etiology and MCs as a potential target for GA.
Materials and Methods
[0045] Human eyes. Human donor eyes were obtained from the National Disease Research Interchange (Philadelphia, Pa., USA). All tissues were obtained within 10-35 hours of death. All donors were Caucasian. GA was diagnosed when a distinct area of RPE degeneration with sharply defined borders without apparent neovascularization. Utilization of the human tissue was in accordance with the Declaration of Helsinki with approval of the Joint Committee on Clinical Investigation at Johns Hopkins University School of Medicine.
[0046] Rats. Sprague/Dawley male rats (200-250 g) were purchased from Envigo (Frederick, Md., USA). MC deficient WsRC.sup.WS/WS rats and their littermate wild-type (WT) WsRC.sup.+/+ rats were purchased from Japan SLC (Hamamatsu, Japan). For all procedures, anesthesia was performed by intramuscular injection of a ketamine (100 mg/mL) and xylazine (100 mg/mL) cocktail. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine eye drops followed by topical anesthesia. All animal experimental procedures were performed according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, following approval from the Animal Care and Use Committee at the Johns Hopkins University.
[0047] Images. Images were captured using Zeiss LSM710 confocal microscope with ZEN software (Carl Zeiss, Jena, Germany), Zeiss Photomic II microscope equipped with QCapture Imaging camera and software (Teledyne QImaging, Surrey, BC, Canada), or Leica AF6000 with Leica Application Suite software (Leica Microsystems, Buffalo Grove, Ill., USA). Images were analyzed using NIH ImageJ (version 1.50), Adobe Photoshop (CS4 and CS6, Adobe Systems, San Jose, Calif.), and Imaris (version 8.3.1, Bitplane USA, Concord, Mass., USA).
[0048] Human flat mount preparation. MCs in human choroidal flat mounts were stained for nonspecific esterase (NSE) and alkaline phosphatase (APase) enzyme activities as previously described..sup.13 Briefly, after the retina was carefully removed, eyecup with choroid was soaked in 1% EDTA (Thermo Fisher Scientific, Waltham, Mass., USA) in distilled water for 2 hours at room temperature (RT) to remove RPE. Any adherent RPE cells were removed by squirting EDTA solution from a syringe with a blunted 25-gauge needle. RPE-denuded choroids were then isolated from the sclera, and fixed for 1 hour in 2% paraformaldehyde (PFA) in 0.1 M cacodylate buffer at RT and incubated for the APase activity as previously described..sup.13 After APase staining, NSE staining was performed using a naphthol AS-D chloroacetate kit (91C-1KT, Millipore Sigma, St Louis, Mo., USA)..sup.13 Granulocytes and MC stained red with this method, but the two cell types were easily distinguished due to their size difference. Then, APase and NSE double stained choroids were postfixed in 2% PFA for 24 hours, washed, and exposed to 30% hydrogen peroxide (Millipore Sigma, St Louis, Mo.) to bleach the melanin at 4° C. as previously reported.13 Total MC number and degranulated MCs at the border of GA and aged matched eyes were counted in five 1 mm.sup.2 areas and averaged per eye for quantification.
[0049] For flat mount IHC choroids, eyes were fixed in 2% PFA in TBS overnight after RPE removal.17 After several washes, choroids were incubated with 5% normal goat serum in 0.1% triton X-100 in TBS with 1% BSA overnight at 4° C., followed by incubation with mouse anti-MC tryptase antibody (Ab)(1:500, ab2378, Abcam, Cambridge, Mass., USA) for 72 hours at 4° C. After washes, the choroid was incubated for 48 hours at 4° C. with goat anti-mouse Ab conjugated with Cy3 (1:200, Jackson ImmunoResearch, West Grove, Pa., USA) and Ulex europaeus agglutinin I (UEA) lectin conjugated with FITC (1:100, GTX01512, GeneTex, Irvine, Calif., USA) and then, imaged with a Zeiss LSM710.
[0050] Rat flat mount preparation. After enucleating the eyes, the anterior segments were removed and the retina was carefully excised. Eyecups were then fixed overnight with 2% PFA at 4° C. After washing, four pie cuts were made to allow flattening of the choroid/sclera eyecup as well as clearly isolate superior and inferior quadrants. Then the choroid was prepared for IHC as previously described..sup.19 The choroid was blocked with 2% normal goat or donkey serum for 4 hours at 4° C., washed in 0.1% triton X-100 in TBS, and then, incubated with a mixture of the following primary Abs: mouse anti-RPE65 (1:200, NB100-355, Novus Biologicals, Centennial, Colo., USA), goat anti-Iba1 (1:200, ab5076, Abcam, Cambridge, Mass., USA), and rabbit anti-MC tryptase (1:200, CAU26568, Biomatik, Wilmington, Del., USA) overnight at 4° C. After washing, they were incubated with Alexa Fluor 488 or Cy3 conjugated goat or donkey secondary Abs (1:300, Jackson ImmunoResearch, West Grove, Pa., USA) overnight, followed by mouse anti-ZO-1 Ab conjugated with Alexa Fluor 594 (1:100, 339194, Thermo Fisher Scientific, Waltham, Mass., USA).
[0051] Rat cross section preparation. Rat eyecups were cryopreserved as reported previously..sup.17 Eight μm cryosections were permeabilized with absolute methanol at −20° C. and blocked with 2% normal donkey serum at RT. After washing, sections were incubated for 2 hours at RT with rabbit anti-MC tryptase Ab (1:200, CAU26568, Biomatik, Wilmington, Del., USA). After washing, sections were incubated for 30 minutes at RT with Isolectin GS-IB4 from Griffonia simplicifolia (GS lectin) conjugated with Alexa Fluor 488 (1:100, 121411, Thermo Fisher Scientific, Waltham, Mass., USA) and donkey anti-rabbit Ab conjugated with Alexa Fluor 647 (1:500, Jackson ImmunoResearch, West Grove, Pa., USA) and then, coverslipped with mounting medium (Vector Laboratories, Burlingame, Calif., USA).
[0052] Hydrogel preparation and injection. A hydrogel which slowly released the compound 48/80 (Millipore Sigma, St Louis, Mo., USA), a snake venomlike compound, was formulated using thiolated hyaluronic acid and 4-arm poly (ethylene glycol) acrylate crosslinked using thiol-ene click chemistry.20 The gel consisted of 48/80 (20 mg/mL), 4-arm PEG-acrylate (100 mg/mL, PSB-421, Creative PEGWorks, Chapel Hill, N.C., USA), 8-arm PEG SH (150 mg/mL, PSB-851, Creative PEGWorks, Chapel Hill, N.C.), hyaluronic acid-SH (200 mg/mL, HA-371, Creative PEGWorks, Chapel Hill, N.C.) added to Irgacure 2959 (Ciba-Geigy, Tarrytown, N.Y., USA). All components were then mixed together by vortex and placed on ice. The final solution was loaded into an insulin syringe with a 31-gauge needle (BD Biosciences, San Jose, Calif., USA) and then, exposed to UV light (AnalytikJena, Upland, Calif., USA) for 1.5 minutes. The release and stability of the hydrogel were evaluated in vitro in PBS, pH 7.4 at 37° C.20. The amount of 48/80 released was measured using bicinchoninic acid (BCA) assay. After confirming the gel formation by its consistency, 30 μL of hydrogel with or without 48/80 (blank hydrogel) was implanted into the superior subconjunctival space.
[0053] Alcian blue staining. After RPE removal, eyecups were fixed in methanol-formalin-acetic acid cocktail for 30 minutes and MCs were further stained with 0.05% Alcian blue (pH 5.0) in 0.02 M sodium acetate buffer for 45 minutes. Choroids were isolated after eyecups were split at the meridian and the number of non-degranulated and degranulated MCs were counted.
[0054] RPE degeneration. After the choroid/sclera eyecup was stained with anti-RPE65 Ab, 4-5 fields from the gel and non-gel areas of each choroid were captured as optimized z-stacks at 10 or 20× magnification with Zeiss LSM710. Images were exported to ImageJ software, converted into 8-bit gray color and then, thresholded. The dark area was considered degenerating RPE. To exclude the area of RPE nucleus (unstained with anti-RPE65 Ab, therefore dark) from the total dark area, particle sizes ranging from 100-infinity (pixels.sup.2) were analyzed so nuclei were excluded. Percentage of RPE degeneration was averaged per choroid and then, quantified.
[0055] Retinal and choroidal thickness. Eight μm cryosections through the optic nerve were stained with Picrosirius Red Stain Kit (Polysciences, Inc, Warrington, Pa., USA) for 5 minutes as recommended by the manufacturer. After rinsing in hydrochloric acid and dehydrating in ethanol, sections were cover-slipped with Permount Mounting Medium (Thermo Fisher Scientific, Waltham, Mass., USA). The sections were then imaged with a Zeiss Photomic II microscope at 10×, and adjacent overlapping images of each section were taken from Ora serrata to Ora serrata. The overlapping images were then stitched using Adobe Photoshop and a panorama image was generated. The entire retina and choroid of each panorama image was precisely hand-traced and areas of the retina and choroid, respectively, were measured using ImageJ.
[0056] For morphologic analyses, eyes were cryopreserved and stained with H&E staining as previously described.sup.15 or fixed with 2.5% glutaraldehyde, 2% PFA in 0.1 M cacodylate buffer. After the anterior segments were excised, eyes were cut in half through the optic disk. Then, eyes were washed and dehydrated and embedded in glycol methacrylate and cut with 2 μm thickness as previously published..sup.21
[0057] Electroretinogram (ERG). After overnight dark adaptation, rats were deeply anesthetized followed by pupil dilation. The rats were placed on a heated platform and full-field scotopic ERGs were elicited by white light flashes with corneal electrodes at intensities ranging from 0.01 to 1 cd.Math.s/m.sup.2 using the Celeris ERG system (Diagnosys LLC, Lowell, Mass., USA). The amplitude of the a- and b-waves was measured from the a-wave trough to the b-wave peak.
[0058] In vitro assays. RBL-2H3 cells (CRL-2256, ATCC, Manassas, Va., USA) were used for the in vitro assay for MC degranulation. Cell degranulation was assessed by the (3-hexosaminidase release as previously described..sup.22 A density of 50 000 cells/well was plated overnight in a 96-well plate. Ten μg/mL of 48/80 or 10 μg/mL of ketotifen fumarate (1(2628, Millipore Sigma, St Louis, Mo., USA)+10 μg/mL of 48/80 dissolved in 100 μL serum free media (DMEM with 1.0 g/L glucose) (Thermo Fisher Scientific, Waltham, Mass.) were added to each well and incubated for 45 minutes. Then, the cells were lysed using 1% triton X-100 to determine MC β-hexosaminidase that was still cell associated. Twenty μL of the supernatant and then, the cell lysate was collected and added to 50 μL of cold 4-Nitrophenyl N-acetyl-B-d-glucosaminide (1.3 mg/mL in Citrate Buffer, pH 4.5, Millipore Sigma, St Louis, Mo., USA) to detect the amount of β-hexosaminidase released. After the plate was incubated for 90 minutes, 100 μL of 0.2 M sodium hydroxide with 0.2 M glycine (pH 10) were added to terminate the reaction. The plate was read at 405 nm wavelength using a microplate reader (ELx808, BioTek, Winooski, Vt., USA). Degranulation was calculated as the percentage of β-hexosaminidase activity measured in the supernatants relative to the total amount of β-hexosaminidase activity in the cells exposed to 1% triton X-100 (100% degranulation).
[0059] ARPE-19 cells (CRL-2302, ATCC, Manassas, Va., USA) incubated in a 24-well plate for 14 days were used for assessing the effect of tryptase on confluent RPE cell monolayer. After washing in PBS, cells were treated with 500 μL serum free media, with or without human lung tryptase (650366-M, Millipore Sigma, St Louis, Mo., USA) (1 or 5 μg/mL) for 72 hours. Then cells were fixed with 4% PFA, permeabilized with 0.1% triton X-100 in PBS, and stained with phalloidin-tetramethylrhodamine B isothiocyanate (1:500, Millipore Sigma, St Louis, Mo., USA). Images of four fields in the center of the well were captured as optimized z-stacks at 10× magnification with Zeiss LSM710. Images were exported to ImageJ software, converted into 8-bit gray color and thresholded. Percentage of the ARPE-19 cell monolayer degeneration was averaged per well and quantified.
[0060] Ex vivo assays. An ex vivo assay was developed to evaluate drugs quickly and efficiently. After enucleation, the anterior segments and retinas were excised and the eyecups were split at the meridian and divided in halves. Then, eyecups were incubated with 300 μg/mL of 48/80 with or without 300 μg/mL of ketotifen fumarate in serum free DMEM in a 48-well plate for 3 hours. To evaluate oral ketotifen fumarate potential to prevent MC degranulation, rats were treated orally twice daily with ketotifen fumarate (15 mg/kg) in PBS or with PBS as control for 4 days and 2 hours prior to enucleation on the 5th day. Eyes were enucleated and exposed in 48/80 serum free DMEM for 3 hours. After washing, RPE was removed and the eyecup fixed for histochemistry. The choroid was isolated from the sclera and MCs were stained for NSE activity and viewed on the Zeiss LSM710 for the autofluorescence of the reaction product. The numbers of non-degranulated and degranulated cells were counted in the whole choroid. MCs were considered as degranulated when they showed irregular shapes or extracellular granules. For WsRC rats, melanin was bleached using the Melanin Bleaching Kits (Polysciences, Inc, Warrington, Pa.) following the manufacture's protocol before proceeding to immunohistochemistry.
[0061] Volumetric and sphericity measurement. Volumetric and sphericity measurements were performed in the rats to analyze changes in shape and size of choroidal macrophages/monocytes stained with Iba1 as previously reported..sup.23 Briefly, randomly chosen 4-5 fields of optimized 40× magnification z-stacks per choroid captured with Zeiss LSM710 were opened in Surpass View of Imaris software and surfaces (surface detail 1.5 μm) were created, using background subtraction (0.5 μm). Voxel number filtering was applied to remove nonspecific particles in the surfaces created. Cells touching others or ones which had only a portion of the cell body visible were omitted from the volume rendering and then, the cell volume and sphericity were quantified with Imaris software.
[0062] Pharmacokinetic study. Ketotifen fumarate quantification in plasma and eye tissue was conducted using high-performance liquid chromatography with tandem mass spectrometry (LC/MS-MS). Briefly, acetonitrile containing 0.5 μM losartan as internal standard was used to extract ketotifen from choroid and retina. Standards were prepared by spiking ketotifen fumarate in naive tissue from 0.003 to 100 μmol/g in a half log dilution series. Samples were weighed and placed in low retention microcentrifuge tubes with 5 μL extraction solution/mg tissue and pestle homogenized. Samples were vortexed followed by centrifugation at 16 000 g for 5 minutes at 4° C. The supernatants were transferred to a 96-well plate and 2 μL were injected for analysis. Samples were analyzed on an UltiMate 3000 UHPLC coupled to Q Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, Mass., USA) using an Agilent EclipsePlus C18 RRHD (1.8 μm; 2.1. 100 mm) column. The mobile phase consisted of water+0.1% formic acid, and acetonitrile+0.1% formic acid. Separation was achieved at a flow rate of 0.4 mL/min using a gradient run. Samples were analyzed in positive ion mode via heated electrospray with capillary temperature set at 350° C. and a spray voltage of 3.5 kV. Data acquisition were performed in selected ion monitoring (SIM) mode isolating 310.1260 m/z (ketotifen fumarate) and 423.1695 m/z (losartan) with a 1 m/z isolation window. Data were acquired and quantified with Xcalibur software (Thermo Fisher Scientific, Waltham, Mass., USA).
[0063] Drug treatments. Rats were pretreated for 1-2 days prior to 48/80 hydrogel injection with either oral treatment of ketotifen fumarate (15 mg/kg) or PBS twice daily. MC tryptase inhibitor APC 366 (5 mg/kg, CAS 258932-85-8, R&D Systems, Minneapolis, Minn.).sup.24 or control vehicle was administered subcutaneously once daily for 8 weeks.
[0064] Statistics. Statistical analyses were performed with Prism 8 (GraphPad Software, San Diego, Calif.) using a 2-tailed Student's t test or one-way ANOVA with a Tukey post hoc comparison test. Data were expressed as mean±SD P values of less than 0.05 were considered statistically significant.
Results
[0065] Sustained MC activation by subconjunctival 48/80-hydrogel implantation in the rat eye. Immunohistochemical localization of tryptase in human GA choroids demonstrated that many choroidal MCs were degranulated at the border of non-atrophic and atrophic choroid (
[0066] Given the distribution of choroidal MCs primarily along the long posterior ciliary arteries in the rat choroid,.sup.1,9 the hydrogel with 48/80 was injected subconjunctivally in Sprague/Dawley (S/D) rats in the superior quadrant of the eye, distant from the midline of eye, and the greatest concentration of MCs (
[0067] Time course after MC degranulation. RPE degeneration associated with MC degranulation was assessed next in S/D rats. Intriguingly, RPE cells stained for RPE65 in whole mount choroids showed degeneration over time following MC degranulation, with significant RPE degeneration and loss after 4 weeks (
[0068] GA-like features lacking in MC deficient rats implanted with 48/80. MC deficient WsRC.sup.ws/ws and their wild-type (WT) littermates (WsRC.sup.+/+) were treated with subconjunctival 48/80-hydrogel to assess whether 48/80 affected only MCs and MC degranulation alone directly contributed to RPE degeneration. WsRC.sup.ws/ws rats carry a defective gene for c-KIT, which is required for MC differentiation..sup.29 MCs were identified with NSE staining of the conjunctiva in WsRC.sup.+/+ rats but not in WsRC.sup.ws/ws rats (
[0069] In vitro and ex vivo assays to evaluate MC degranulation and drug efficacy. Given that MCs contributed to the GA-like changes in the rat model, it appeared that MC degranulation could be a therapeutic target and, therefore, quiescing MCs might prevent this phenotype. To this end, the therapeutic potential of a MC stabilizing drug was assessed first by co-incubating the established RBL-2H3 rat MCs with ketotifen fumarate, a MC stabilizer, with or without 48/80 and analyzing the degranulation response using a β-hexosaminidase release assay, as previously reported..sup.22 Ketotifen fumarate is a second-generation non-competitive histamine H-1 receptor antagonist. The mechanism of stabilizing MCs is assumed to be by blocking the intracellular calcium channels needed to form granules..sup.32 It has been used to treat conditions such as asthma, allergic conjunctivitis and mastocytosis..sup.33-35 Ketotifen fumarate treatment in the in vitro assay yielded a 56% inhibition of β-hexosaminidase activity (
[0070] Ketotifen fumarate and tryptase inhibitor as therapeutic drugs. To assess whether ketotifen fumarate could prevent GA-like changes in vivo in the present inventors' rat model, pharmacokinetic (pK) evaluation of oral ketotifen fumarate was performed first. Rats were given 3 mg/kg of ketotifen fumarate in PBS or PBS as control once daily by gavage until sacrifice. The animals for pK were sacrificed on the 5th day and the retina and choroid were dissected from the globes. The pK evaluation indicated that there was 13-29 nM ketotifen fumarate in choroid and 12-52 nM in retina. Knowing that the drug reached choroid, 15 mg/kg of ketotifen fumarate in PBS was administered twice daily by gavage, while controls received PBS orally. Pretreatment of rats with ketotifen fumarate for 4 days prior to enucleation prevented 37% of MC degranulation and significantly prevented macrophage activation in the ex vivo assay (
[0071] Tryptase is the most abundant protease released by MCs' and tryptase release was increased in choroid/Bruch's membrane of GA subjects..sup.17 A similar increase in tryptase release was observed in the rat model (
[0072] Therefore, the tryptase inhibitor APC 366, which is reported to prevent allergic, inflammatory or fibrotic changes in human and animal models.sup.24,38,39 was evaluated in the present inventors' 48/80-hydrogel implant rat model. Rats were given a subcutaneous injection of APC 366 (5 mg/kg) or vehicle daily after 48/80 implantation until sacrifice. After 4 weeks treatment, RPE degeneration was significantly inhibited (
Discussion
[0073] GA is a multifactorial disease characterized by RPE loss and choriocapillaris attenuation and retinal and choroidal atrophy that is clinically manifested over many years. There is increasing evidence that hypoxia-initiated oxidative stress and inflammation contribute to GA pathogenesis..sup.40 Nevertheless, there is insufficient understanding of the cellular or molecular mechanism underlying the development of GA, as well as, few animal models reproducing a GA-like phenotype..sup.12 This paucity of knowledge and animal models has hampered the drug development and targeting. In the current study, the present inventors present evidence that chronic degranulation of choroidal MCs can induce many phenotypic changes in retina and choroid that occur in GA: RPE degeneration, visual function decline, and retinal and choroidal thinning. However, another hallmark of GA, attenuation of choriocapillaris,.sup.21 did not occur in the present inventors' model, perhaps due to the regenerative capability of the rodent choriocapillaris..sup.41 Furthermore, the present inventors developed both ex vivo and in vivo models, which demonstrate that choroidal MC degranulation alone can stimulate choroidal macrophage activation as observed in human GA..sup.23 Using these models, the present inventors demonstrated that MC stabilization and tryptase inhibition can prevent these MC-induced GA-like phenotypes. MCs are the initial responders of innate immunity and their abundance in the uveal tract and choroid suggest their fundamental importance in the eye..sup.1 Indeed, they respond to toxins and microbes as well as substances found in GA choroid such as AGEs, C3a, C5a, and CRP, all of which are implicated in AMD..sup.14,15,42,43 Recently, the present inventors reported that choroidal MCs were increased in number and their degranulation was increased in GA subjects..sup.13 The present inventors do not know exactly when abnormal MC degranulation occurs in GA choroid, but in that study the present inventors found a significant increase in MC numbers and number of degranulated in early AMD subjects, suggesting it is occurring early in the disease process. In addition, MC-derived tryptase was localized in Bruch's membrane..sup.13,17 Nevertheless, the precise role of MCs in the development of GA had yet to be elucidated. Here, the present inventors demonstrate that hydrogel containing 48/80 implanted subconjunctivally resulted in a gradual and protracted activation and degranulation of choroidal MCs in the rat. Continuous MC degranulation mediated chronic macrophage activation and induced RPE degeneration with subsequent retina and choroidal thinning, four characteristics of human GA..sup.21,23 The present inventors further show evidence that MC-derived tryptase, at least in part, through degradation of the choroid stroma and Bruch's membrane, contributed to RPE degeneration. The pathological phenotype was prevented by genetically eliminating MCs and by pharmacological intervention targeting MCs with a generic MC stabilizer ketotifen fumarate and a MC specific tryptase inhibitor APC 366. Therefore, MCs with aberrant degranulation and specifically MC-derived tryptase appear to play a central role in developing a GA-like pathology. Therefore, the present inventors propose the hypothesis that choroidal MC degranulation and release of MC-derived tryptase (
[0074] To evaluate the efficacy of targeting MCs in GA, development of an assay to screen potential therapeutic compounds was deemed indispensable. To this end, the present inventors utilized the well-established rat MC line, RBL-2H3 cells, and their production of β-hexosaminidase, an MC granule enzyme.
[0075] Although RBL-2H3 cells have been broadly used to assess MC degranulation and drug screening, various contradictions and limitations have also been reported..sup.36 There remains controversy in the lineage of RBL-2H3 cells in that they resemble basophils rather than MCs, which is highly likely because these cells originate from a basophilic leukemia..sup.36 The ex vivo assay developed for these studies overcame those limitations and provided a rapid assay in a tissue milieu involved in AMD, the choroid and its inflammatory cells. This assay enabled sufficient choroidal MC degranulation and effective drug screening in 3 hours and yielded data on three important parameters: percentage of MCs degranulated in choroid, and volume and sphericity of choroidal macrophages, indicating macrophage activation. The lack of blood flow enabled direct evaluation of only choroidal MCs and resident macrophages and not interaction with circulating cells. In order to exclude the possibility of 48/80 influencing macrophages directly and not activating MCs specifically, MC deficient WsRC.sup.WS/WS and their WT control rats were first evaluated in this ex vivo assay. Choroidal macrophages were only activated in WT, not in MC deficient rat choroids, suggesting cytokines released after MC activation and degranulation-induced macrophage activation. Similar findings were confirmed in vivo, collectively indicating that cytokines released from activated MCs induced focal inflammation and contributed at least in part to the disease progression. However, the present inventors realize that the present inventors' model only represents MC interaction with macrophages and macrophages in AMD choroid are exposed to an activated complement cascade and CRP as well as MC granule contents.
[0076] In the current study, the pharmaceutical benefit of ketotifen fumarate, an FDA-approved generic MC stabilizer, was evaluated in vitro, ex vivo, and then, in vivo, where it prevented MC degranulation efficiently in all experiments. One of the major complications reported with oral daily treatment was drowsiness but this is said to decrease with time..sup.33 Patients with mastocytosis and chronic asthma are managed chronically with oral MC stabilizers like ketotifen fumarate without severe complications, suggesting that the drug is rather innocuous..sup.44,45 However, a recent report demonstrated that ketotifen fumarate dose dependently decreased non-rapid eye movement (REM) sleep and increased REM sleep in rats; therefore, it might function as an aid for desirable sleep..sup.46 Nevertheless, no adverse effects with oral ketotifen fumarate treatments were observed in the present inventors' study.
[0077] Inhibiting MC-derived tryptase activity with APC 366 successfully inhibited all GA-like changes in the present inventors' rat model from RPE degeneration to macrophage activation after MC degranulation. Degeneration of RPE and retinal and choroidal thinning was expected because tryptase degrades collagens and activates MMPs, which further degrade stroma and basement membranes. Previous reports have shown that tryptase released via MC degranulation amplified the production of intercellular adhesion molecule-1, CC-motif ligand 2, and IL-8 in endothelial and epithelial cells..sup.47,48 Most recently, treating mice with APC 366 suppressed inflammation as assessed by the transcriptional expression of multiple mediators including IL-1β, IL-6, IL-8, CC-motif ligand 2, and MMP-3 in an osteoarthritis model..sup.49 Thus, the effect of tryptase released in degranulation resulting in macrophage activation might well be expected in the present inventors' model. Also, MCs release IL-6 and tumor necrosis factor when activated, further stimulating macrophage activation..sup.49 Taken together, monocyte/macrophage activation in the choroid and activation of resident macrophages was presumably prevented by inhibiting tryptase with APC 366, hence inhibiting focal inflammation. The present inventors have clarified a critical role of MCs in RPE degeneration and the presence of MC-derived pro-inflammatory cytokines and proteases can be presumed to contribute to disease development based on other studies..sup.38,47 One scenario might be that MCs are activated by stressed RPE pro-inflammatory molecules in the GA choroid and then, catalyze tissue destruction via tryptase and also trigger inflammation and macrophage activation, which facilitates the vicious cycle of degeneration in the GA photoreceptor/RPE/Bruch's membrane/choriocapillaris complex (
[0078] In conclusion, this is the first study to demonstrate the involvement of MC degranulation and release of tryptase in initiating GA-like pathologic changes in the eye. It would be interesting in the future to investigate if there is any relationship between incidence of GA and MC activating diseases like mastocytosis, MC activating syndrome, Ehlers-Danos syndrome, and fibromyalgia. The etiology of RPE degeneration and choroidal thinning in GA was not known prior to this study. The present inventors' model suggests a potential role for MCs in GA without addressing the pro-inflammatory milieu of GA RPE/choroid complex, where complement is activated and oxidative stress is present. A MC stabilizer or inhibition of MC-derived tryptase prevented the disease onset and progression in the rat model. These results support the novel concept that MCs contribute to the development of GA and choroidal MCs could be a viable therapeutic target for the currently untreatable GA.
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