Dorzolamide Nanoparticle Loaded Contact for Treatment of Glaucoma BMED 4751 ­ Intro to Biomaterials

Professor Krishnendu Roy April 26, 2016

Julie Bu jbu7@gatech.edu Madison Lewis mlewis44@gatech.edu Lianhua Shen lshen44@gatech.edu Meghan Styles mstyles@gatech.edu

PHS 398 Research Plan SPECIFIC AIMS Glaucoma is a disease that damages the optic nerve due to increased pressure caused by fluid in the eye1. By 2020, it is estimated that 79.6 million people will have open angle and angle closure glaucoma. Bilateral blindness will be present in 11.2 million of this glaucoma afflicted group2. Glaucoma is the second leading cause of blindness worldwide and can begin developing at age 40. Current treatments include daily eye drops, oral medication, and various surgeries1. Eye drops are the most common form of treatment and must be used 1­3 times a day3, depending on the particular medication, at regular intervals to decrease the amount of fluid produced or to increase fluid drainage. Eye drop treatments include prostaglandin analogs, beta­blockers, alpha adrenergic agonists, carbonic anhydrase inhibitors, and miotics4. Carbonic anhydrase inhibitors (CAI) are most common for oral medication and lower intraocular pressure (IOP) by decreasing aqueous production by direct antagonist activity on the ciliary epithelial carbonic anhydrases5. Oral carbonic anhydrase inhibitor medication is usually used in conjunction with eye drops to reduce fluid production. Laser trabeculoplasty and conventional surgeries help fluid drain out of the eye, but may cause other problems for the patient by resulting in cataracts1. As the older population in the U.S. grows, so does the prevalence of diseases of aging such as glaucoma. In order to help these men and women fight this disease caused by overproduction of aqueous humor, our project seeks to design and test a biomaterial contact lens in a comprehensive fashion that will increase compliance by reducing dose frequency and wearer interaction. Previous studies have shown that PAA/amine terminated 4­Arm PEG (A4PEG) nanoparticles are effective in ophthalmic drug release. However, studies have not been conducted to calculate the ideal PAA/A4PEG ratio necessary to create a zero­order release kinetic of dorzolamide, a carbonic anhydrase inhibitor, when bound to contact lenses. Given these findings, we hypothesize that contact lenses embedded with PAA­A4PEG nanoparticles loaded with dorzolamide will be effective at treating glaucoma. This hypothesis will be addressed in the experiments of the following Specific Aims:

Specific Aim 1. To determine the ratio of PAA­A4PEG nanoparticles that can effectively 1) diffuse through the cornea and reach the ciliary body 2) demonstrate near zero­order dorzolamide drug release kinetics in aqueous humor Specific Aim 2. To determine whether the immunological reaction in response to the drug loaded contact lenses is significant. Specific Aim 3. To test whether attached PAA­A4PEG NPs interfere contact lens visual functionality

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RESEARCH STRATEGY (a) SIGNIFICANCE Glaucoma is the second leading cause of blindness worldwide. It is regarded as a priority eye disease by WHO6, with currently over 12% of the global blind population having become blind as a result of this condition. The disease is hard to prevent, but can be mitigated if diagnosed early and treatment is properly maintained in these early stages. As the population of the US grows older and lives longer as a result of medical technologies, so does the prevalence of diseases of aging such as glaucoma. In order to help these men and women fight glaucoma resulting from overproduction of aqueous humor, this project seeks to design a daily biomaterial contact lens that improves drug delivery by controlling the timing and location of drug release and improving compliance by reducing the dose frequency. The proposed work will provide an important new tool for delivering a glaucoma drug and may open up a nanoparticle delivery method for a variety of drugs needed to be delivered into the eye. (b) INNOVATION Many of the current treatments pose problems for proper treatment maintenance and product efficacy. The most common treatment for glaucoma, eye drops, requires multiple daily applications which may result in low compliance. In addition, the medication is washed away from the precorneal area 100 times faster than it can penetrate the cornea due to tears7. Generating a sustained drug release method requires knowledge of ratios of PAA and n­amine PEG required for diffusion. This project will apply experimental testing to measure the diffusivity, drug release kinetics, and contact lens functionality of our biomaterial to provide new information on ocular drug release methods. One particular treatment, TRUSOPT®, is administered with one drop in each affected eye three times a day3. This drug warns against wearing contacts during administration and up to 15 minutes after drops are put in due to its use of benzalkonium chloride, which may be absorbed by soft contact lenses. During this time the patient is without contacts and may have trouble seeing or may have to switch to glasses until they can reinsert their lenses. The wearer must take their contacts in and out multiple times, also leading to lower compliance due to inconvenience. Having a daily, slow drug­releasing contact with equivalent refraction and functionality to the user’s usual prescription lenses will encourage proper use without having to remove your prescription lenses and only requiring one administration in the morning. In ongoing experiments, drug loaded contacts for the treatment of diseases such as glaucoma are being tested on their way to the American pharmaceutical market. Most of these lenses are soaked soft contacts or drops placed onto lenses with a release rate only lasting a few hours after soaking, so the wearer must repeatedly put in more drops or resoak the lens8. More recently, embedded nanoparticles are being studies as a drug transport mechanism in contact lenses. Some of these particles rely on enzyme degradation of an outer coating containing the drug9,10. We hope to add to this research with a different, more simple approach to a drug loaded nanoparticle using PAA­A4PEG. (c) APPROACH Preliminary Studies. We propose to test the hypothesis that our contact will release dorzolamide at an appropriate rate for the treatment of glaucoma while retaining the non­immunogenic response and functionality of typical contacts. The basis for this hypothesis is founded by the precedent set by the PAA­PEG models demonstrated by Vasi. Additional testing is needed to prove that the novel combination of hydrogels will not elicit any adverse immune response or other effects in the wearer and will still allow the user to see by maintaining appropriate transparency and refractive index. In sum, the above research indicates that drug particles in a hydrogel medium could be used for treatment of diseases such as glaucoma. At this point, we have very little information about how our particular drug and hydrogel combination will perform. It is also not

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clear what improvements could be made until further testing is done. The experiments listed in this proposal are designed to explore these questions. Specific Aim 1. To determine the ratio of PAA/A4PEG nanoparticles that can effectively 1) diffuse through the cornea and reach the ciliary body 2) demonstrate near zero­order dorzolamide drug release kinetics in aqueous humor Hypothesis. We anticipate that this study will reveal that PAA­A4PEG nanoparticles loaded with dorzolamide will diffuse through the cornea and reach the ciliary body (kcornea 10­6) and demonstrate near zero­order≥ dorzolamide drug release kinetics. Rationale. In this study, nanoparticles will be fabricated by PAA and A4PEG through polymer crosslinking. PAA is the polymer that exhibits very high adhesive bond strength in contact with tissues, enhancing mucosal penetration of drugs. Furthermore, they allow the localization of the drug at the absorption site to aid the sustainable long­term release of the drug. A4PEG is a hydrophilic, nonionic, and biocompatible polymer for pharmaceutical and biomedical applications. Its hydrophilicity exhibits excellent solubility both water and organic media. Such a property will aid contact lenses to increase hydration to the eyes and add comfort for users while wearing them. In addition, its non­immunogenicity will reduce the risk of eye irritation and inflammation. The morphology and surface charge is strongly dependent on the molar ratio of PAA­A4PEG and the PAA concentration. A study confirmed that formulation of PAA­A4PEG molar ratio with 0.3% wt PAA concentration is the optimum condition to obtain desired degree of swelling and hydrophilicity of the matrix. Hence, nanoparticles will be fabricated based on the given ratios. Dorzolamide will be loaded in the nanoparticles to test the swelling property, and the kinetics of drug loading and releasing. Moreover, cornea diffusivity coefficient is calculated based on the average molecular weight from the obtained product and octanol­water distribution coefficient. Experimental Approach. PAA­A4PEG Nanoparticles Preparation A4PEG has four arms that are terminated by amine groups. These branched arms will react with carboxylic group in PAA to form nanomer size and spherical shaped nanoparticles. In ideal process, carboxylic group in PAA will interact with the all of the 4 arms of the A4PEG to form the crosslinking (Figure 1). In order to improve the reaction efficiency EDAC will be added as the catalyst. EDAC is a carboxyl activating agent to activate coupling of amine group and carboxylic group. First, PAA (0.3% wt) will be mixed with EAC with molar ratio : PAA/EDAC = ¼, at 5, under vigorous stirring (1000 rpm) to form an emulsified mixture. After emulsification, aqueous solution of PEG (0.3% wt) is added dropwise in the mixture under the stirring with molar ratio: PAA:PEG = 3:1, for 24 hours at 4­7C11. The obtained product will be washed with hexane and stirred for 5 days with deionized water. Ultimately, they will be freeze­dried to remove all the remaining water, and desired nanoparticles will be achieved.

In Vitro Dorzolamide Release Kinetics PAA­A4PEG Nanoparticles(10 mg) will be immersed in an aqueous solution of dorzolamide (5 ml; 2%,w/w,pH 4.012). They will be stirred for 24 hours at room temperature to ensure the maximum saturation of the dorzolamide diffusion. Afterward, 10 mg drug loaded nanoparticles will be suspended in 5 ml of phosphate buffer and sealed in a dialysis membrane. NanoDrop ND 1000 Spectrophotometer will be used to record the drug release11. We will also run bank experiments with drug­free nanoparticle as control group to confirm there is no interfering background adsorption. Dorzolamide is hydrophobic drug. And combination of PAA and

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A4PEG forms non­biodegradable hydrophilic nanoparticles. Thereby, drug will diffuse through corea by its affinity towards hydrophobic cell membranes. Cornea Diffusivity Coefficient The overall permeability of the cornea (kcornea) is determined by the series combination of the resistance to transport the three tissues, epithelium, stroma and endothelium, respectively13.

Equation 1 is a theoretical model that was developed by Edwards and Prausnitz. They were able to estimate the physicochemical properties of the cornea and diffusing solutes and predict the passive, steady­state permeability of cornea to a broad range of compounds. In this study, we will implement this method to calculate the corneal permeability to dorzolamide. Initial estimates of corneal permeability can be made knowing only the following two parameters: 1) molecular radius, which can be determined using established correlations based on molecular weight and/or chemical structure and 2) octanol–water distribution coefficient, which can be measured experimentally or calculated using semi­empirical correlations for octanol–water partition coefficient and degree of ionization13. In Vivo Sustained Release Fluorescence Tracking PAA­A4PEG nanoparticles will be labelled with the near infrared fluorophore AlexaFluor® (using Thermo Fisher Scientific’s SAIVI™ Rapid Antibody Labeling Kit)14. Fluorescently tagged PAA­A4PEG concentrations will be administered through the diffusion into rabbit cornea. Non­invasive near infrared imaging of the pancreas will detect the fluorescence14. The image will go through a code in MATLAB to count the number of fluorescent pixels and turn it into a percentage of the image which can be compared with the initial concentration. Interpretation of Results. After PAA­A4PEG nanoparticles are prepared, Gel Permeation Chromatography will be conducted to assure all the nanoparticles are of relatively the same size. After dorzolamide is loaded to the nanoparticles, we anticipate to observe drug release pattern in two stages with initial burst followed by a sustained release through diffusion (Zero­order kinetics). In addition, a prior study indicated that corneal permeabilities were calculated to be in the region between 10­5 to 10­7 cm/s13. Therefore, we expect to obtain corneal permeability faster than the average high value, which is 10­6 cm/s to assure drug­loaded contact lenses can effectively pass the nanoparticles through the cornea and reach the target site ­ ciliary body. PAA­A4PEG nanoparticles are reported to be non­biodegradable. However, the eyes are connected to the nose and mouth through lacrimal punctum. Thereby, we expect that the fluorescently tagged nanoparticles will be reduced from ciliary body region. We also anticipate to observe that nanoparticles will drain into the lacrimal punctum further into the nasolacrimal duct, and eventually into the backside of the nose and mouth to enter the digestive system. From there they will be degraded by the stomach and eliminated as waste. Potential Problems and Alternative Approaches. Should the results of our initial study not match the diffusion rate we had anticipated, corneal diffusion rate can be altered by adjusting molar ratio of PAA to A4PEG. More A4PEG would increase hydrophilicity and would increase water interaction, while having a higher ratio of PAA would decrease this property.

Specific Aim 2. To determine whether the immunological reaction in response to the drug loaded contact lenses is significant. Hypothesis: The body’s immunological reaction in response to the biomaterial shall not be significantly different than non­drug loaded contact lenses.

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Rationale. The human immune system is poised to generate a response upon interaction with a foreign body, the nanoparticles. However, the eye presents a unique phenomenon known as ocular immune privilege. This privilege is the eye’s way of maintaining vision by mediating and limiting local immune responses. Experimental Approach. An experiment will measure cytotoxicity of various PAA­A4PEG concentrations by measuring cell metabolic activity. To do this we will be using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay®11. Collected human dermal microvascular endothelial cells (HDMEC) will be seeded into a 24 well­plates and then exposed to varying concentrations of PAA­A4PEG nanoparticles. The cells will be exposed for 24 and 48 hours. After these time points the cells will be washed with phosphate buffered saline (PBS) and incubated in a medium containing the MTS solution from the CellTiter 96® for one and a half hours. After this the cells will be transferred and the optical density will be measured photometrically at 492 nm in an ELISA 96 well­plate reader. The results obtained will be in terms of the average of vilability the standard deviation. The± Vasi paper obtained results showing little damper to proper cell function when performing these tests (Figure 211). Testing the rabbit eye with PAA­A4PEG nanoparticles will also show the possible immune response in a human eye. We can look for the expression of CD68 which is a glycoprotein that is expressed by humans and is useful as a marker for various cells in the macrophage lineage. Immunohistochemistry (IHC) can be used for the detection of CD68 in rabbits. After PAA­A4PEG nanoparticles are added to a cell culture and incubated for 24 and 48 hours, an ELISPOT will be used to measure cytokine activity15. Interpretation of Results. We are hypothesizing that the nanoparticles will not create an active immune response. The differences between the chromatograms at different time points are not significantly different. If not, a time point to reach steady state can be found. Potential Problems and Alternative Approaches. A potential problem could arise if significant immune response is detected. Though this is not expected, it is always a possibility. The addition of an immune suppressant could help if needed. Specific Aim 3. To test whether attached PAA­A4PEG NPs interfere contact lens visual functionality Hypothesis: The drug­loaded contact lenses preserve the transparency and refractive index to retain visual functionality. Rationale. While patients use the drug­loaded contact lenses, they will not be able to use corrective lenses that contact the eye. Therefore, if a patient needs to wear the drug­loaded contact lenses, the biomaterial needs to retain the visual functionality of a daily contact lens. Experimental Approach. Comprehensive analyses will be carried out on contact lenses created with and without drug­loaded nanoparticles.

1. Refractive index. ASTM D542, Standard Test Method for Index of Refraction of Transparent Organic Plastics, shall be used to calculate the index of refraction to the nearest significant figure warranted by the accuracy and duplicability of the measurement for both control and experimental groups. A minimum of three specimens of each group shall be prepared and measured. The test specimens shall be of a size that will conveniently fit on the face of the fixed half of the refractometer prisms. A source of

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diffuse polychromatic light will be used. The temperature in degrees Celsius at which the index was measured.

2. Transparency. ASTM D­1003, Standard Test Method for Haze and Luminous Transmittance of Transparent Plastics, shall be used to calculate the light transmission percentages for both control and experimental groups. A minimum of three samples will be prepared to test each group.

Interpretation of Results. We anticipate that the index of refraction between control and experimental groups will not be significantly different. In addition, the index of refraction and light transmittance transmittance for the experimental group shall be within the tolerances as stated in ISO 18369 for ophthalmic optics in contact lenses. Therefore, contact lenses with the PAA­A4PEG dorzolamide loaded nanoparticles will retain visual acuity. Potential Problems and Alternative Approaches. Given existing data on PAA­A4PEG nanoparticles, the drug loaded contact lenses should be transparent. Since the nanoparticles are attached as a coating to existing contact lenses, refractive index should not change. Should the findings suggest that visual acuity is affected, we will shift our attention to changing the concentration of nanoparticles attached to lenses. In addition, we will look into attaching the nanoparticles to only the skirt of the lenses. Therefore, there will be no coating directly on the corneal area, and the patient will be able to freely see without obstruction. VERTEBRATE ANIMALS

1. Rabbit studies. New Zealand albino rabbits are requested for the project. 2. Justification of animal use. Animals must be used because we are examining the effect of the contact

lens on the eye as an organ. There are no artificial eyes to represent both the immunological functions and the glaucoma of the eye in vitro. Numbers of animals were justified in section 1.

3. Veterinary care. Veterinary care is provided by staff, which carries out a variety of laboratory diagnostic procedures and provides oversight for preventive health quality assurance program. Services are provided either “in­house”, or are referred to outside laboratories as necessary. Necropsy and histopathology can be performed according to investigator needs, from basic or comprehensive diagnostic studies to tissue­specific or comprehensive research studies. Complementary clinical pathology procedures may be included as part of the comprehensive work­ups. Clinical pathology work can also be provided, including: (1) clinical hematology, including: complete blood counts, differential white blood cell counts, reticulocyte counts, platelet counts, hematocrit; (2) serum biochemistry; (3) parasitology; (4) cytology, (5) microbiology; (6) urinalysis, (7) diagnostic serology for specific animal pathogens, (8) molecular biology for specific animal pathogens.

4. Humane treatment. Animals undergoing more than momentary distress will be anesthetized using standard protocols (isoflurane, buprenorphine, ketamine/xylazine). Subsequent to any invasive procedure, animals will be monitored until recovery from anesthesia, and at least daily for the first week thereafter, for adverse symptoms including hunched posture, loss of hair, weight loss, lethargy, or other symptoms of abnormal appearance or behavior. Mice will be treated as recommended by the attending veterinarian, or if more appropriate, euthanized to relieve severe discomfort.

5. Method of euthanasia. Euthanasia will be performed by carbon dioxide asphyxiation followed by thoracotomy. This method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

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BIBLIOGRAPHY AND REFERENCES CITED 1. National Eye Institute. Facts About Glaucoma. NIH National Eye Institute. 2. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. British Journal of Ophthalmology. 2006;90(3):262­7. doi: 10.1136/bjo.2005.081224. 3. Trusopt [package insert]. Whitehouse Station, NJ: Merck & Co., Incorporated; 2014. 4. Haddrill M, Slonim C. Glaucoma Treatment: Eye Drops and Other Medications. All About Vision. April 2016. 5. Jindal A. Medical Management for Primary Open Angle Glaucoma. American Academy of Ophthalmology. October 2015. 6. World Health Organization. Priority Eye Diseases. Prevention of Blindness and Visual Impairment. 7. Plazonnet B. Ophthalmic Drug Delivery In Modified­Release Drug Delivery Technology. 2003;289­313. doi:10.1201/9780203910337.pt3 8. Hu X, Hao L, Wang H, Yang X, Zhang G, Wang G, Zhang X. Hydrogel Contact Lens for Extended Delivery of Ophthalmic Drugs. International Journal of Polymer Science. 2011;2011:9. doi: 10.1155/2011/814163. 9. Kim H­J, Zhang K, Moore L, Ho D. Diamond Nanogel­Embedded Contact Lenses Mediate Lysozyme­Dependent Therapeutic Release. ACS Nano. 2014;8(3):2998­3005. doi: 10.1021/nn5002968. 10. Mukherjee B, Shekhar Dey N, Maji R, Bhowmik P, Jyoti Das P, Paul P. Current Status and Future Scope for Nanomaterials in Drug Delivery, Application of Nanotechnology in Drug Delivery. InTech. 2003; doi: 10.5772/58450. 11. Vasi A­M, Popa MI, Tanase EC, Butnaru M, Verestiuc L. Poly(Acrylic Acid)–Poly(Ethylene Glycol) Nanoparticles Designed for Ophthalmic Drug Delivery. Journal of Pharmaceutical Sciences. 2014;103(2):676­86. doi: 10.1002/jps.23793. 12. Wilson CG. Topical drug delivery in the eye. Experimental eye research. 2004;78(3):737­43. Epub 2004/04/27. PubMed PMID: 15106953. 13. Edward A, Prausnitz M. Predicted permeability of the cornea to topical drugs. Pharm Res.2001;18(11), 1497­1508. 14. ThermoFisher Scientific. Small animal in vivo imaging antibody labeling. 2016. 15. My Bio Source. Anti­CD68 antibody :: Rabbit anti­Mouse, Rat CD68 Polyclonal Antibody. 2016. 16. Brubaker R. F. The flow of aqueous humor in the human eye. Transactions of the American Ophthalmological Society. 1982;80, 391­474. 17. Glaucoma Research Foundation. Glaucoma Facts and Stats. 2015. 18. Inoue J, Oka M, Aoyama Y, Kobayashi S, Ueno S, Hada N, . . . Takehana M. Effects of Dorzolamide Hydrochloride on Ocular Tissues. Journal of Ocular Pharmacology and Therapeutics. 2004; 20(1).

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19. Leese H, Bhurtun V, Lee KP, Mattia D. Wetting behaviour of hydrophilic and hydrophobic nanostructured porous anodic alumina. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2013;420, 53­58. doi:10.1016/j.colsurfa.2012.12.010 20. Mayo Clinic Staff. Glaucoma: Treatments and Drugs. 2015.

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