Thiolated poly(aspartic acid) polymers in ophthalmic therapy Gabriella Horvát1, Benjámin Gyarmati2, Barnabás Szilágyi2, András Szilágyi2, Szilvia Berkó1, Erzsébet Csányi1, Piroska Szabó-Révész1, Giuseppina Sandri3, Maria Cristina Bonferoni3, Carla Caramella3, Mária Budai-Szűcs1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Szeged, Szeged, Hungary 2 Soft Matters Team, Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budapest, Hungary 3 Department of Drug Science, Faculty of Pharmacy, University of Pavia, Pavia, Italy Dear ….I would like to present the benefits of the Thiolated poly(aspartic acid) polymers in ophthalmic therapy. But first let me introduce the importance and the problematics of local ophthalmic therapy. 5th International Conference and Exhibition on Pharmaceutics & Novel Drug Delivery Systems March 16-18, 2015 Crowne Plaza, Dubai, UAE

Problems with current local ocular drug delivery systems Drug dilution and removal within minutes: blinking, baseline and reflex lachrymation Slow absorption of drugs: anatomy, physiology, barrier function of the cornea Frequent instillations of eye drops: to maintain a therapeutic drug level in the tear film or at the site of action toxic side effects, cellular damage at the ocular surface The topical ocular drug delivery systems are considered to be the safest, least invasive, and most self-administrable dosage forms. Unfortunately, there are many problems with the current local ocular formulations on the market. The blinking, baseline and reflex lachrymation lead to fast dilution and removal of the drug formulation. If the drug is able to remain on the ocular surface throughout the diluting and draining experienced by the tear film, it then faces its next barrier the cornea which increases the absorption time. To maintain the therapeutic drug level frequent instillations of the eye drops are needed amounting to toxic side effects and cellular damage at the ocular surface. All these processes and barriers result in a very low bioavailability, which is less than 5%, even after frequent applications of the formulations. VERY LOW BIOAVAILABILITY (<5%) Ludwig, A., Adv. Drug Deliver. Rev. 2005, 57, 1595-1639

Challenges in ocular drug delivery formulation Anatomical Special anatomy Protective mechanisms Limited area for absorption Biopharmaceutical Hydro - and lipophilicity Molecule size Protein bindings Enzymatic degradation of the drugs In case of ocular drug delivery formulation many factors have to be taken into consideration, such as anatomical, biopharmaceutical and patient driven challenges. The greatest barrier to ocular drug delivery is the anatomy of the eye itself, its protective mechanisms, such as blinking and lachrymation, and also the limited area for absorption. The biopharmaceutical challenges include the hydro - and lipophillicity, the molecule size, the protein bindings and the enzymatic degradation of the drugs. And last but not least the patients’ needs have to be respected, such as few applications, easy handling and (self)-administration, no discomfort or foreign body sensation, blurring vision, no visual disorders, to be non-invasive and also have a low price. Patient Few applications (two or less) Easy handling and (self)-administration, No discomfort or foreign body sensation, blurring vision No visual disorders Be non-invasive Low price

New possibility in ophthalmic therapy Mucoadhesive polymers take advantage of the ocular surface mucosal layer. Mucoadhesion is adhesion between the drug delivery system and the mucosa. Physical and chemical interactions can occur during mucoadhesion. Methods for the investigation of mucoadhesion: Rheology: determination of the changes in rheological parameters, after mixing the mucoadhesive polymer with mucin. Tensile test: Adhesive strength is defined as the external force required for the separation of the two interfaces. The work of adhesion (A, mN mm) can be calculated as the area (AUC) under the “force versus distance” curve. One way to overcome some of the eye’s natural anatomical barriers is to take advantage of the ocular surface mucosal layer and to formulate a drug delivery system with mucoadhesive properties. Mucoadhesion is an important phenomenon in ocular formulation, which is the adhesion between the drug delivery system and the mucosa. During mucoadhesion physical interactions, like the interpenetration of the polymer chains into the mucin layers, and chemical bond formation between the entangled chains can occur. The use of mucoadhesive polymers is leading to the increase of residence time of the drug at the ocular surface, drug uptake, diffusion and transport, while the concentration, required volume and usage frequency can be decreased. There are several methods for the investigation of mucoadhesion, but the two most common are the rheological and the tensile test measurement.

Thiolated poly(aspartic acid) polymers Synthetized by Soft Matters Group of Budapest University of Technology and Economics Thiol containing side groups bonded to poly(aspartic acid) Biocompatible and biodegradable polymers Non-toxic and do not generate immunogenicity Redox sensitive, in situ gelling Optically clear solution and gel In our research work thiolated poly(aspartic acid) polymers were characterized, synthetized by the Soft Matters Group of Budapest University of Technology and Economics. Thiol containing side groups were bonded to poly(aspartic acid). These polymers are biocompatible and biodegradable, non-toxic and do not generate immunogenicity. They are redox sensitive and in situ gelling. Their solutions and gels are optically clear. B. Gyarmati, B. Vajna, Á. Némethy, K. László, A. Szilágyi., Macromol. Biosci. 2013, 13, 633–640

Aims Assign the effects of the mucin on the gelation time Characterize the mucoadhesion of the ThioPASP polymers Define the function of the thiol groups in the mucoadhesion Determine the drug release profile The goals of our work were to determine the in situ gellation and to assign the effects of the mucin on the gelation time. Characterize the mucoadhesion of the ThioPASP polymers and define the function of the thiol groups. Since the ThioPASP polymers were planned to be used in ocular therapy, measurements were performed to determine the release profile and the kinetics of the active ingredients from the gels. Horvát, G., Gyarmati, B., Berkó, Sz., Szabó-Révész, P., Szilágyi, B. Á., Szilágyi, A., Soós, J., Sandri, G., Bonferoni, M.C., Rossi, S., Ferrari, F., Caramella, C., Csányi, E., Budai-Szűcs, M., Eur. J. Pharm. Sci. 67, 1-11 (2015)

In situ gelation with and without mucin No gelation below 10%w/w ThioPASP Addition of mucin aided the gelation The gelation time was shorter with mucin The rate of gelation and final value of storage modulus increased in the presence of mucin During rheological, in situ gellation, measurements no gelation was observed below 10%w/w ThioPASP concentration. Then we added mucin to the samples and measured the gelation time again. The addition of mucin aided the gelation. The gelation time was shorter and the final value of storage modulus increased in the presence of mucin. It can be established that probably in in vivo conditions shorter gelation time can be expected.

Mucoadhesion – Rheology Mucin augmented the elastic modulus Physical and chemical interactions between the polymer and the mucin The added mucin decreased the slope of the curves Mucoadhesion of the ThioPASP polymers was first determined by the rheological method. As we can see mucin augmented the elastic modulus indicating that interactions occurred between the polymer and the mucin. In our samples the added mucin decreased the slope of the curves suggesting the formation of a chemically cross-linked structure between the polymer and mucin chains in addition to physical entanglement.

Mucoadhesion – Tensile Test Measurements were made both with mucin dispersion (8%) (in vitro) and with blank (simulated lachrymal fluid) „A” increased continuously as the concentration was elevated „F” reached a maximum at 7% w/w polymer The chemical bonds have a larger effect at lower polymer concentration (increasing F) At high polymer concentration, the potential for chemical bonds reached a maximum (the free thiol groups were saturated at the interface - plateau of F) „A” did not reach a maximum because of the increasing interpenetration. (More cross-links resulting in a gel structure, which induces increased swelling, allowing deeper and improved interpenetration) probe sample We also investigated the mucoadhesion of ThioPASP polymers by the tensile test. The sample was placed on the probe and put in contact with mucin dispersion or lachrymal fluid. The value of work of adhesion increased continuously as the concentration was elevated, while the force reached a maximum at 7% w/w polymer concentration. According to the results it can be established that the chemical bonds have a larger effect at lower polymer concentration, but at high polymer concentration, the potential for chemical bonds reached a maximum, because the free thiol groups were saturated at the interface. This is the explanation why the force curve reached a plateau. While work of adhesion did not reach a maximum thanks to the increasing interpenetration. At higher concentration more cross-links were formed resulting in a gel structure, which induced increased swelling, allowing deeper and improved interpenetration. Mucosa or mucin disp.

Mucoadhesion- “Wash away” “Wash away‟ ex vivo measurements mimic the lachrymation of the eye, under conditions relatively close to real mucoadhesive circumstances of the eye. Model drug: sodium fluorescein (0.008%) Increase of the ThioPASP concentration was accompanied by a slight decrease in the amount of model drug being washed away. In the reference systems the observations were similar. ThioPASP formulations have a longer residence time (40-50% vs. 0-30% w/w). ThioPASP “Wash away‟ ex vivo measurements mimic the lachrymation of the eye, under conditions relatively close to real mucoadhesive circumstances of the eye. Modified Franz diffusion cell was used, where simulated lachrymal fluid was streamed through the donor chamber. The measurements were made on excised porcine conjunctiva which was placed on the acceptor chamber. As a model drug sodium fluorescein was used. Increase of the ThioPASP concentration was accompanied by a slight decrease in the amount of model drug being washed away. This was observed also in the reference systems. According to the results ThioPASP formulations have a longer residence time on the surface of the conjunctiva, 40-50% vs. 0-30% w/w. HEC

Drug release kinetics I. Model drug: sodium diclofenac (0.01%) First hour: fast diffusion of the SD ThioPASP polymers have a high water uptake → they have a lower cross-linking density, and the SD is therefore able to diffuse through this structure more easily In the drug release measurements as a model drug, sodium diclofenac was used. In the first hour a fast diffusion of the SD can be observed. This is very beneficial, because the therapeutic drug level can be reached. These polymers have a swelling-controlled drug release mechanism so we determined the swelling properties of the ThioPASP polymers. The ThioPASP has a high water uptake, suggesting a lower cross-linking density, and this is why SD is therefore able to diffuse through this structure more easily.

Drug release kinetics II. Drug release kinetics according to Korsmeyer-Peppas equation: 𝑀 𝑡 𝑀 ∝ =𝑘 𝑡 𝑛 where 𝑀 𝑡 𝑀 ∝ is the fraction of drug released, k is the kinetic constant and n is the release exponent describing the mechanism of the release Swelling exponents (n): 0.874 Release exponents (n): 0.6561 We also determined the kinetic constant and the release exponents according to Korsmeyer- Peppas equation in drug release and swelling measurements. As a result it can be established that we have a swelling controlled non-Fick diffusion of sodium diclofenac from the ThioPASP gels, because the release exponent is between 0.5 and 1.   During the drug release, our polymer gels placed on the membrane continuously swelled, which led to a simultaneous absorption of the water and desorption of drug via a swelling-controlled diffusion mechanism. This combination of diffusion, swelling, and relaxation is responsible for the non-Fickian release mechanism. Non-Fick diffusion 0.5 ˂ n ˃ 1

Mucin exhibited a strong effect on cross-link formation Conclusion Mucin exhibited a strong effect on cross-link formation Faster in situ gelation Strong mucoadhesion Resistance against lachrymation Appropriate drug release profile We can conclude that mucin exhibits a strong effect on cross link formation of the ThioPASP gels, leading to a faster in situ gelation and strong mucoadhesion, which is beneficial in ocular therapy, because they can be easily used as eye drops and after the instillation they will in situ gellify. Thanks to their strong mucoadhesion they are resistant against lachrymation which was proved by the wash away test. They also have an appropriate drug release profile with a first fast drug release followed by a sustained release of the encapsulated drug for up to 24 h. The results proved the importance of the presence of the thiol groups and suggested that a ThioPASP formulation can be useful as an in situ gelling, ocular mucoadhesive drug delivery system. ThioPASP polymers can be potential in situ gelling, ocular mucoadhesive drug delivery system

Acknowledgements Dr. András Szilágyi, Ph.D Benjámin Gyarmati Cooperation partners: Soft Matters Group, Budapest University of Technology and Economics Dr. András Szilágyi, Ph.D Benjámin Gyarmati Barnabás Szilágyi Department of Drug Sciences, University of Pavia Prof. dr. Carla Caramella, D.Sc. Prof. dr. Maria Cristina Bonferoni, Ph.D., Dr. Giuseppina Sandri,Ph.D. Department of Pharmaceutical Technology, University of Szeged Prof. Dr. Pharm. Piroska Szabó-Révész, D.Sc. Dr. Pharm. Erzsébet Csányi, Ph.D. Dr. Pharm. Mária Budai-Szűcs, Ph.D. I would like to thank our cooperation partners from the Soft Matters Group, Budapest University of Technology and Economics for providing the thiolated poly(aspartic acid) polymers for my research work; to our cooperation partners from the Department of Drug Sciences, University of Pavia for giving me the opportunity to do the tensile test measurements at their department. And I would also like to thank my colleagues from the Department of Pharmaceutical Technology, University of Szeged for all their help.

THANK YOU FOR YOUR KIND ATTENTION! Thank you for your kind attention and if you have any questions I will gladly answer them.