CT Imaging and Scaling

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Intravitreal Pharmacokinetics in Mice: SPECT/ CT Imaging and Scaling to Rabbits and Humans Mechthild Schmitt, Eero Hippeläinen, Manuela Ravina, Blanca Arango-Gonzalez, Maxim Antopolsky, Kati-Sisko Vellonen, Anu J. Airaksinen, and Arto Urtti Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00679 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Molecular Pharmaceutics

Intravitreal Pharmacokinetics in Mice: SPECT/CT Imaging and Scaling to Rabbits and Humans 1

Mechthild Schmitt, 2,3 Eero Hippeläinen, 1 Manuela Ravina, 6 Blanca Arango-Gonzalez, 1 Maxim Antopolsky, 5 Kati-Sisko Vellonen, 4Anu J. Airaksinen, 1,5,7 Arto Urtti* 1

Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 2 University of Helsinki, Department of Physics, P.O. Box 64, FI-00014 University of Helsinki 3HUS Medical imaging Center, Clinical physiology and nuclear medicine, University of Helsinki and Helsinki University Hospital, Finland 4 Department of Chemistry - Radiochemistry, University of Helsinki, Helsinki, Finland 5 School of Pharmacy, University of Eastern Finland, Kuopio, Finland 6 Centre for Ophthalmology, University Eye Hospital Tübingen, Tübingen, Germany 7 Laboratory of Biohybrid Technologies, Institute of Chemistry, St. Petersburg State University, Peterhoff, Russian Federation Correspondence: *Arto Urtti, Ph.D. Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy University of Helsinki, Viikinkaari 5 E, POB 56, 00790 Helsinki, Finland [email protected] tel: +358 40 540 2279 Graphical abstract

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Preclinical in vivo tests of retinal drug responses are carried out in mice and rats, often after intravitreal injections. However, quantitative pharmacokinetics in the mouse eye is poorly understood. Ocular pharmacokinetics studies are usually done in the rabbits. We investigated elimination of three compounds ([99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin with molecular weights of 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively) from mouse vitreous using imaging with single photon emission computed tomography / computed tomography (SPECT/CT). Increasing molecular weight decreased elimination of the compounds from the mouse eyes. Half-lives of [99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin in the mouse eyes were 1.8 ± 0.5 h, 4.3 ± 1.7 h and 30.0 ± 9.0 h, respectively. These values are 3-12 fold shorter than half-lives of similar compounds in the rabbit vitreous. Dose scaling factors were calculated for mouse-to-rabbit and mouse-to-man translation. They were 2790 and 38-126, respectively, for intravitreal injections in the rabbit and man. We show ocular pharmacokinetic parameters for mice and inter-species scaling factors that may augment ocular drug discovery and development. Key words: SPECT/CT imaging; intravitreal injection; pharmacokinetics; mouse; retinal drug delivery; human translation; molecular weight; inter-species scaling Introduction Intravitreal injection is a widely used clinical method in retinal and choroidal drug delivery. This approach is used because topical eyedrops and systemic administration do not provide adequate delivery of drugs to the retina. Intravitreal injections are commonly used to treat neovascular and edematous conditions in the posterior eye segment with corticosteroids and anti-VEGF agents (monoclonal antibodies, Fab-fragments, soluble receptors) 1,2. Overall, 5.9 million intravitreal anti-VEGF injections were given in the USA in 2016. 3 Intravitreal drugs with adequate membrane permeability are eliminated from the vitreous via posterior route, i.e. across the blood-retina barriers to the systemic blood circulation 4. Impermeable compounds (e.g. biologicals) are eliminated mostly via anterior route by diffusing in the vitreous to the posterior chamber and, thereafter, by elimination within the outflow of aqueous humor 4,5. Therefore, anti-VEGF compounds have long half-lives in the vitreous (from days to about one week), whereas the half-lives small molecules, capable of crossing the bloodretinal barrier, are usually less than 10 hours 6,7,8. Intravitreal pharmacokinetics is usually studied in rabbits and occasionally in man 9. The volume of drug distribution in the rabbits is nearly constant and similar as the volume of the vitreous humour (about 1.5 ml), but vitreal clearance values differ over 100-fold range, depending mostly on drug permeability in the blood-retinal barrier and drug elimination via anterior route 6-9. Consequently, intravitreal half-lives of injected compounds show a wide range in the rabbit vitreous (1-144 hours) 6. Intravitreal clearance values in the rabbit and human eyes correlate well, enabling pharmacokinetic translation to man: human clearance (ml/h) = 1.41 x rabbit clearance (ml/h) + 0.04 9. Unfortunately, inter-species scaling factors do not exist for rodent-to-man translation. Preclinical efficacy of experimental retinal drug treatments are usually tested with mice and rats 10-12. The mouse models (incl. transgenics) for ophthalmic diseases are economical,


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ethical and widely available, whereas rabbits are only rarely used to test retinal drug efficacy. Unfortunately, pharmacokinetic studies in the mouse vitreous have not been published in peerreviewed journals. Therefore, drug responses and drug concentrations in the mouse eye cannot be linked with each other and rational scaling of drug doses from mice to rabbits or humans is not possible. Intravitreal pharmacokinetic data and parameters for mouse are needed in ophthalmic drug research and development. Ocular pharmacokinetic studies in mice are challenging, because the volume of adult mouse vitreous is only 5 µL. Some inherited pathologies are investigated in young mice with even smaller vitreal volumes. Only a few studies have reported drug concentrations in the mouse eyes after intravitreal injections 13-15. Fluorescent imaging of tissue slices has been performed more widely, but this approach is qualitative and limited only to fluorescent compounds 16,17. Quantitative pharmacokinetic data for intravitreal injections into the mice are needed for scaling from mouse to man. In this study, we used single positron emission computed tomography/computer tomography (SPECT/CT) for quantitative imaging of intravitreally injected radiolabeled materials in mouse eyes. We present relationship between the molecular weight and clearance of intravitreal substances from mouse eyes. Furthermore, we calculated pharmacokinetic scaling factors based on the SPECT/CT mouse data and prior information about intravitreal kinetics in rabbits and man. Materials and methods [99mTc]Tc-pentetate (PENTACIS®, CIS bio international, Gif sur Yvette, France), [111In]In-pentetreotide (Octreoscan®, Mallinckrodt Medical B.V, Le Petten, The Netherlands) and [99mTc]Tc-human serum albumin (Vasculocis®, CIS bio international, Gif sur Yvette, France) were prepared from commercially available labelling kits. 99mTcO4- eluted from a MEDIGEN-Tc® generator (MAP Medical Technologies) was used. Molecular weights of the [99mTc]Tc-pentetate, [111In]In-pentetreotide and [99mTc]Tc-human serum albumin were 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively. Animal studies were reviewed and approved by FELASA, the Finnish National Animal Experiment Board and all procedures were performed according to the ARVO statement for the use of animals in ophthalmic and visual research. Male C57BL/6JRccHsd mice (26-35 g) aged 810 weeks were purchased from Envigo (Horst, The Netherlands) and housed under standard day/night lighting circles with free access to food and water. A total of 9 animals were divided into 3 groups with n=6 eyes per group. Each group was intravitreally injected with one of the three test compounds. Prior to the intravitreal injections, the mice were intravenously anaesthetized with 0.05 ml/10 g of a medetomidine-ketamine anesthesia solution that was diluted from Domitor® vet 1 mg/ml (Orion Pharma, Espoo, Finland), Ketaminol® vet 50 mg/ml (Intervet International GmbH, Germany) and 0.9% saline solution. The final doses of medetomidine and ketamine were 1 mg/kg and 75 mg/kg, respectively. Prior to the intravitreal injections, the mice received local anaesthetic (oxybuprocaine hydrochloride eyedrops; Oftan® Obucain 4 mg/ml, Santen Pharmaceuticals Ltd., Tampere, Finland) and after injections topical fucidic acid eye gel (Flucithalmic VET 1 %, Dechra Pharmaceuticals PLC, Upplands Väsby, Sweden) to prevent any bacterial infections.



The intravitreal injections were performed under a microscope using the sharpened tip of a glass capillary which was connected to a 10 µl Hamilton syringe. A small incision in the eye bulb was made below the ora serrata where 1 µl of radiolabeled test compound was injected into each mouse eye. The needle was maintained in the eye for about 3 seconds to avoid any efflux of the injected compound. All eyes received 0.2 – 2.5 MBq of radioactivity. SPECT/CT imaging was performed using a preclinical four-headed gamma camera outfitted with multi-pinhole collimators having in total 36 of 1.0 mm pinholes and a CT scanner (Nucline® NanoSPECT/CT, Bioscan Inc., Washington, DC, USA; manufacture and maintenance by Mediso Ltd., Budapest, Hungary; collimators from Scvis GmbH, Göttingen, Germany). The sensitivity of the pinhole collimators is >1200 counts per second (cps)/MBq which gives a maximum resolution of ≤ 0.75 mm. CT imaging was carried out with 45 kV tube voltage in 180 projections. The scanning mode of both SPECT and CT is helical. While scanning, all animals were continuous anaesthetized by an inbuilt isoflurane system (Scanbur AB, Sollentuna, Sweden) with a set flow of 0.5 – 1.5 % isoflurane (Vetflurane, 1000 mg/g, Virbac, Carros, France) in oxygen. The body temperature of the animals was maintained using a heated animal bed (Equipement Vétérinaire Minerve, Esternay, France). SPECT images of the eyes were collected in a set of dynamic images containing 6 scans with 20 projections and 240 seconds/projection resulting in a total acquisition time of 2 hours. The following time points, one static scan of 30 minutes with 16 projections and 450 seconds / projection, were acquired. After each SPECT scan, a CT scan was taken to overlay the images of SPECT and CT to provide the anatomical reference next to the radiotracer data of SPECT. The images were reconstructed with HiSPECT NG software (Scivis GmbH, Göttingen, Germany). The reconstructed images were overlaid and further analyzed with the help of Vinci program (Max Planck Institute for Metabolism Research, Cologne, Germany). Elliptical volumes of interest were defined around the vitreous humour and the aqueous humour to follow the vitreal elimination of the test compounds. Decay correction was done for all dynamic data. Physical half-lives of 6.01 hours and 67.32 hours were used for Tc-99m and In-111 (nucleide.org), respectively, to carry out exponential decay correction to all time dependent data in the study. Due to the decay correction, the curves in graphs represent amount of the pharmaceuticals in the eyes as a function of time. The resolution of SPECT/CT did not allow precise differentiation of the radioactivity in the vitreal cavity and anterior chamber. Therefore, they were treated as joint single compartment. The elimination rates of the labelled materials from the eyes were solved with Phoenix 64 software (Pharsight, Certara, USA) using curve fitting. One compartmental model for bolus injection was used. The optimal weighing schemes were as follows: [111In]In-pentetreotide (uniform weighing), [99mTc]Tc-pentetate (1/yfitted weighing) and [99mTc]Tc-human serum albumin (1/yfitted weighing). The results are presented as mean ± standard deviation. Results The three test compounds with different molecular weights showed different rates of elimination from the eye (including both vitreous and anterior chamber) (Fig. 1). The rate of elimination decreased with increasing molecular weight. Based on the pharmacokinetic curve fitting the half lives in the mouse eye were 1.8 ± 0.5 h for [99mTc]Tc-pentetate, 4.3 ± 1.6 h for [111In]In-pentetreotide and 30.0 ± 9.0 h for [99mTc]Tc-human serum albumin (Table 1). The first-


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order rate constants for elimination were 0.4062 ± 0.0942 h-1 for [99mTc]Tc-pentetate, 0.1842 ± 0.0756 h-1 for [111In]In-pentetreotide, and 0.02544 ± 0.0099 h-1 for [99mTc]Tc-human serum albumin.

Figure 1. Radioactivity of ([99mTc]-pentetate (A), [111In]-pentetreotide (B) and [99mTc]-human serum albumin (C) as a function of time in mouse eyes (n=6-8). Table 1. Pharmacokinetic parameters of radiotracers in the mouse eyes. PK parameter [99mTc]-pentetate [111In]Inpentetreotide Half-life (h) 1.8 ± 0.5 4.3 ± 1.6 -1 Elimination rate constant (h ) 0.4062 ± 0.0942 0.1842 ± 0.0756 Clearance (µl/h) 2.0 0.9 Mean residence time (h) 2.5 5.4 Volume of distribution (µl) 5.0 5.0 Molecular weight 510.2 1506.4

[99mTc]-human serum albumin 30.0 ± 9.0 0.02544 ± 0.0099 0.13 39.2 5.0 66,500

Mouse eyes were too small for differential quantitation of radioactivity in the vitreous and anterior chamber. However, the images in Figure 2 show the presence of [99mTc]-pentetate, [111In]In-pentetreotide and [99mTc]-human serum albumin both in the vitreous cavity and anterior chamber at various time points. Fusion of SPECT and CT images for [99mTc]-pentetate injection immediately after injection and at 41 minutes also demonstrate redistribution of the tracer in the mouse eyes.



Figure 2. Example SPECT time series of [93Tc]Tc-pentetate, [111In]In-pentetreotide, and [93Tc]Tc-albumin elimination after intravitreal injection in mouse eyes. The compounds distribute partly from the vitreous to the anterior chamber over time. Anterior segment (‘Ant’) and posterior segment (‘Post’) have been marked in the pictures at time zero (i.e. immediately after the injection).

Figure 3. SPECT-CT fusion pictures of mouse head 0 minutes (A) and 41 minutes (B) after [93Tc]Tc-pentetate injection. The figure shows deep scanning through mouse eyes.


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Discussion Elimination of intravitreally injected compounds from mouse eye seems to depend on the molecular weight: the elimination slows down with increasing molecular size. In mice, the ocular half-life showed about 17-fold range from [99mTc]Tc-pentetate (molecular weight 510.2 Da) (1.8 h) to [99mTc]Tc-human serum albumin (30 h) (molecular weight 66.5 kDa). Human serum albumin is hydrophilic and large molecule, and it is not expected to pass through the tight blood ocular barriers. The other test compounds are also hydrophilic based on calculated logD7.4 values without technetium or indium (pentetate -7.36 and pentetreotide -1.76). The hydrophilic compounds are expected to show low permeability in the blood-ocular barriers 6. Therefore, significant elimination via anterior route is expected and shown as radioactivity in the mouse anterior chamber after the intravitreal injections (Fig. 2). However, [99mTc]Tc-pentetate and [111In]In-pentetreotide may eliminate to some extent also through the blood retinal barrier. In addition, they [99mTc]Tc-pentetate and [111In]In-pentetreotide may be eliminated faster than [99mTc]Tc-human serum albumin via anterior route that is controlled by the diffusion in the vitreous, vitreal dimensions and the accessible route from the vitreous to the aqueous humor 18,19. These routes were demonstrated and modeled in previous rabbit studies, but the exact parameter values and models for the mice are missing. In rabbit eyes, the scale of ocular half-lives is 26-fold for hydrophilic compounds with similar molecular weight scale (from ≈ 500 to ≈ 60 000) and, again, the large molecules have longer ocular half-lives than the smaller compounds. Furthermore, the ocular half-lives of the hydrophilic (logD7.4 -2.29 to -9.54) compounds with molecular weights of 454-554 were 5.6-23.6 hours in the rabbits 6,20,21; i.e. 3.1-12.3 times longer than the half-life of [99mTc]Tc-pentetate (molecular weight 510.2 Da) in the mouse eyes. The ocular half-life of vancomycin (logD7.4 = 4.49) in the rabbit vitreous was 25.89 h 6,22: about 6 times longer than the ocular half-life of [111In]In-pentetreotide (4.3 h) in mouse eyes. In rabbits, pegaptanib (molecular weight 50,000 Da) and sEphB4 (molecular weight 57,800 Da) had ocular half-lives of 91.6 h and 143.6 h, respectively 6,23,24. The half-lives in the rabbit eyes are 3.1-4.8 times longer than the ocular halflife of intravitreal albumin in mouse eye (30 h). Overall, the half-lives in mouse eyes seem to be 3-12 times shorter than in the rabbit vitreous. The scaling factors of the half-lives have been compiled in Table 2. Del Amo et al. 6 determined the apparent volumes of distribution for 52 intravitreal compounds in the rabbit. Interestingly, the values were close to the anatomical volume of the rabbit vitreous for all compounds (lipophilic and hydrophilic, small and large). Since our test compounds are hydrophilic, they are not expected to bind or partition significantly into the ocular cells. In addition, drug binding to the vitreal macromolecules is modest 25. The injection volume (1 µl) increases the vitreal volume maximally to 6 µl, but this volume is expected to return rapidly back to normal by water resorption from the vitreous and possible spill-over after the injection. Therefore, we use anatomical volume of the mouse vitreous (5 µl) to estimate ocular clearance of the compounds (equation 1). CL = k x V

Equation 1

In Eqn. 1 k is the elimination rate constant (determined values; h-1) and V is the vitreal volume (5 µl) 26. The following clearance estimates were obtained for intravitreal compounds in mice: 2 µl/h for [99mTc]Tc-pentetate, 0.9 µl/h for [111In]In-pentetreotide, and 0.13 µl/h for [99mTc]Tc-



human serum albumin (Table 1). Like in rabbits, the clearance values are smaller than the rate of aqueous humor outflow (10.8 µl/h in mouse eyes) 27, because the transfer of hydrophilic compounds to the anterior chamber is substantially hindered by the physical barriers of the iris, ciliary body and lens 18. The estimated clearance values in mice are about 27-90 times lower than the reported vitreal clearance values in the rabbits (11-179 µl/h) for hydrophilic compounds with similar scale of molecular weights.6 Drug exposure is described as the area under the concentration vs time curve (AUC). Dependence of AUC on the dose and clearance (CL) is shown in equation 2: AUC = Dose / CL

Equation 2

Clearance in rabbits is 27-90 times higher than in the mice. Therefore, 27-90 times higher doses are required in rabbits to reach the same vitreal drug exposure (AUC) than in the mice. Vitreal clearance in man is 1.4 times greater than in the rabbits 9, resulting in the dose scaling factors of 38-126 for mouse-to-man translation. The dose scaling values for small and large molecules are illustrated in Table 2. Table 2. The proposed pharmacokinetic scaling factors for mouse-to-rabbit and mouse-to-man translation. The scaling factors are presented to small hydrophilic compounds and macromolecules based on the results of this study. The scaling factors for small lipophilic compounds are based on the blood retinal barriers surface areas in the species. Mouse-to-rabbit scaling factor Mouse-to-man scaling factor Parameter Small Small Macromolecule Small Small Macromolecule lipophilic hydrophilic lipophilic hydrophilic Half-life 10 3-12 3-5 10 5-36* 5-15* Clearance 32 27-90 84 45 38-126 118 Dose 32 27-90 84 45 38-126 118 *Scale factor from Del Amo et al.7 was used to convert half-life scaling values of mouse-to-rabbit to mouse-to-man values.

The clearance estimates guide also the selection of intravitreal dosing levels in mouse eyes. In Equation 3, effective drug concentrations from in vitro studies can be used to estimate the relevant target AUC values. In Equation 3, AUC = Css,av x 

Equation 3

Css,av = average steady state concentration in the vitreous (i.e. effective concentration from in vitro studies) and  = dosing interval. Required dosing can be further calculated with clearance and AUC (Equation 2). The clearance values (Table 1) can be used as guidance in the intravitreal dose estimation in mouse studies. Our SPECT/CT imaging instrument has spatial resolution of ≤ 0.75 mm. Even though imaging shows qualitatively drug distribution over time into the anterior part of the mouse eyes (Figs 2-3), this resolution was not adequate for reliable and distinct tracer quantitation in the vitreal cavity and anterior chamber. Therefore, we pooled the radioactivity of the posterior and anterior segments and used these values for the kinetic calculations. This approach should represent the rate of elimination from the vitreous for the following reasons. Firstly, the concentrations of intravitreally injected large molecules in the aqueous humor are only about


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10% or less of the concentrations in the vitreous in the rabbits 28,29. We do not know the quantitative distribution posterior vs anterior eye segment in the mice. Secondly, the concentrations of biologicals in the aqueous humor reflect drug elimination from the vitreous, because the vitreal elimination controls the access of drug to the aqueous humor in the rabbits 28. The situation in mice should be similar, because the aqueous humour outflow (10.8 µl/h) is 5-83 times higher than the measured ocular clearance values (0.13-2.0 µl/h). This indicates that overall ocular elimination is controlled by drug decay from the vitreous cavity. Due to the smaller dimensions in the mouse eyes, it is likely that the intravitreal drugs will distribute faster to the mouse anterior chamber than to the rabbit or human anterior chamber. We used hydrophilic radiolabeled compounds in this study and the results may not be directly applicable to lipophilic small molecules that are rapidly eliminated through blood retinal barrier. In general, lipophilic drugs should be eliminated faster from the mouse eye than [99mTc]Tc-pentetate suggesting higher clearance values than 2 µl/h and shorter half-lives than 1.8 h in mouse eyes. Vitreal clearance of lipophilic compounds is determined by the surface area of the blood retinal barrier and its permeability 6,7. If we assume similar blood retinal barrier permeability for mouse and rabbit eyes, the clearance values should be related to the ratio of the surface areas of these barriers in mouse (16.5 mm2) and rabbit (520 mm2). Thus, mouse-to-rabbit scaling factor would be 32; not different from the range obtained for hydrophilic compounds (2790). The scaling factor from mouse to man is 1.4 times higher (about 45) (Table 2). The elimination rate constant (k), and half-life, are related to clearance and volume of distribution (k = Cl/V; t1/2 = ln2 V/CL) (Equation 1). Then, we can estimate that the half-lives of posteriorly eliminating lipophilic compounds are 10 times longer in rabbit than in mouse, which is in line with the half-life difference for hydrophilic compounds (3-12 fold). This study shows for the first time quantitation of intravitreal compounds in mouse eyes and presents their kinetic elimination parameters. We performed this study with SPECT/CT imaging, but this method and the instrument are expensive. Furthermore, the method requires labeling of the test compounds prior to imaging, which limits the available chemical space. For these reasons this method is not widely applicable for ocular pharmacokinetic studies. However, the kinetic parameters and scaling factors can be widely used pharmaceutical and ophthalmological research and development. The main factors of intravitreal pharmacokinetics in mice are the same as in the rabbits and humans. Conclusion We studied the elimination of intravitreal compounds from mouse eyes using SPECT/CT imaging. Our data show clear relationship between the molecular weight and ocular elimination in mouse eyes. The values of half-life and clearance are smaller in mouse than in rabbit and human eyes, but we estimated dose scaling factors for translation of drug doses from mouse to rabbit and man. The study will also give guidance for the dose selection in mouse experiments after in vitro potency characterization of new drug candidates. Acknowledgements Funding from Academy of Finland to Anu Airaksinen (136805) and Arto Urtti (311122) is acknowledged. Imaging was done at the nanoSPECT/CT laboratory, Faculty of Pharmacy, University of Helsinki, part of the Helsinki Institute of Life Science (HiLIFE) and supported by



Biocenter Finland. Manuela Ravina was supported by the Government of Spain research visit grant. Arto Urtti was also supported by Government of Russian Federation Mega-Grant 14.W03.031.0025 “Biohybrid Technologies for Modern Biomedicine”. References 1. Holz FG, Tadayoni R, Beatty S, Berger A, Cereda MG, Cortez R, Hoyng CB, Hykin P, Staurenghi G, Heldner S, Bogumil T, Heah T, Sivaprasad S. Multi-country real-life experience of anti-vascular endothelial growth factor therapy for wet age-related macular degeneration. Br. J. Ophthalmol. 99, 220-226 (2015). 2. Mitchell P, Liew G, Gopinath B, Wong TY. Age-related macular degeneration. Lancet 392, 1147-1159, 2018 3. Grzybowski A, Told R, Sacu S, Bandello F, Moisseiev E, Loewenstein A, SchmidtErfurth U. Update on Intravitreal Injections: Euretina Expert Consensus Recommendations. Ophthalmologica 239:181–193 (2018). 4. Maurice DM, Mishima S. Ocular pharmacokinetics. In: Sears M (Ed.) Handbook of Experimental Pharmacology. Springer-Verlag, Berlin-Heidelberg, pp. 16-119. 5. Araie M, Maurice DM. The loss of fluorescein, fluorescein glucuronide, and fluorescein isothiocyanate dextran from the vitreous by the anterior and retinal pathways. Exp. Eye Res. 52, 27-39, 1991. 6. Del Amo E, Vellonen KS, Kidron H, Urtti A. In Silico Prediction of Intravitreal Primary Pharmacokinetic Parameters and Drug Concentrations: Tool for Ocular Drug Development. Eur. J. Pharm. Biopharm. 95, 215-226, 2015. 7. Del Amo E, Rimpelä AK, Heikkinen E, Kari OK, Ramsay E, Lajunen T, Schmitt M, Pelkonen L, Bhattacharya M, Richardson D, Subrizi A, Turunen T, Reinisalo M, Itkonen J, Toropainen E, Casteleijn M, Kidron H, Antopolsky M, Vellonen KS, Ruponen M, Urtti A. Pharmacokinetic Aspects of Retinal Drug Delivery. Progr. Retin. Eye Res. 57: 134185, 2017. 8. Kim HM, Park KH, Chung JY, Woo SJ. A Prediction Model for the Intraocular Pharmacokinetics of Intravitreally Injected Drugs Based on Molecular Physicochemical Properties. Ophthalmic Res. 21:1-9, 2019 9. Del Amo E, Urtti A. Rabbit as an animal model for intravitreal pharmacokinetics: Clinical predictability and quality of the published data. Exp. Eye Res. 137, 111-124, 2015. 10. Trifunović D, Arango-Gonzalez B, Comitato A, Barth A, Sahaboglu A, del Amo EM, Kulkarni M, Hauck SM, Ueffing M, Urtti A, Arsenijevic Y, Marigo V, Paquet-Durand F. HDAC inhibition protects degenerating cone photoreceptors in vivo. Hum. Mol. Genet. 25, 4462-4472, 2016. 11. Shah M, Cabrera-Ghayoni S, Christie LA, Heid KS, Viswanath V. Translational preclinical pharmacological disease state models for ophthalmic drug development. Pharm. Res. 36-58, 2019 12. Rappoport D, Morzaev D, Weiss S, Vieyra M, Nicholson JD, Leiba H, GoldenbergCohen N. Effect of intravitreal injection of bevacizumab on optic nerve head leakage and retinal ganglion cell survival in a mouse model of optic nerve head crush. Invest. Ophthalmol. Vis. Sci. 54, 8160-8171, 2013.


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