J Vis Exp. 2016; (113): 53878. Seong Joon Ahn, 1 , 2 , 3 Hye Kyoung Hong, 1
Young Mi Na, 1 Sang Jun Park, 1 , 2 Jeeyun
Ahn, 1 , 2 , 4 Jaeseong Oh, 5 Jae Yong Chung, 6
Kyu Hyung Park, 1 , 2 and Se Joon Woo 1 , 2 The intraocular route of drug
administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G
needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as
the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs. Keywords: Medicine, Issue 113, drug, eye, intraocular, intravitreal, pharmacokinetics, rabbit Before the advent of intraocular drug delivery, the main concern of medical therapy for intraocular diseases was the efficiency with which the drug could
penetrate into the eye. The blood-ocular barrier prevents many substances, including drugs, from diffusing into the eye. Therefore, concentrations of drugs that are above therapeutic levels may not be easily obtained. The intraocular drug administration method, including intracameral and intravitreal injections, can directly bypass the blood-ocular barrier1-3, so that therapeutic concentrations of drugs can be achieved in the eye4,5. Accordingly, intravitreal
drug delivery has become a popular method of treatment for several intraocular diseases5,6. For example, intravitreal injection is widely performed for age-related macular degeneration, diabetic retinopathy, retinal vein occlusions, and intraocular infections7-10. In particular, since the introduction of anti-VEGF medications, the frequency of intravitreal injections has remarkably increased for the treatment of retinal diseases. Therefore, it is important to understand the
intraocular pharmacokinetics of such drugs for evaluating the efficacy and safety of the medical therapy. Although the intraocular administration of drugs is considered a major breakthrough in medical therapy for ocular diseases, monitoring the drug concentration within the eyeball is technically demanding. Because human eyes contain only small amounts of aqueous humor (about 200 µl) and vitreous (about 4.5 ml, Table 1), it is technically difficult to obtain
sufficient amounts of ocular fluid to measure the drug concentration. Furthermore, methods that are used to obtain the eye fluid, such as vitreous tapping or anterior chamber paracentesis, may damage the ocular tissue and result in serious complications, such as cataracts, endophthalmitis, or retinal detachment11,12. Accordingly, animal models are used in pharmacokinetic studies of commonly used intraocular drugs13. Among these animal models, rabbits or monkeys are the most
frequently used animals. Rabbits, which are small mammals of the order Lagomorpha in the family Leporidae, are found in several parts of the world. Because rabbits are not aggressive, they are easy to handle, use in an experiment, and observe. Lower cost, ready availability of the animal, similar eye size to humans, and a large database of information for comparison favor performing pharmacokinetic studies using rabbit eyes. In this paper, a protocol for pharmacokinetic studies of
intraocular drugs in rabbit eyes is described. Our protocol follows the guidelines of the Institutional Animal Care and Usage Committee (IACUC) of Seoul National University Bundang Hospital, which approved all of the animal procedures and animal care methods presented in this protocol. The IACUC is in full compliance with the eighth edition of Guide for the Care and Use of Laboratory Animals (2011). All procedures were
performed with adherence to the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research in animals. Individual cages were used for housing the rabbits. Additional surgery or preparation before carrying out this experiment (i.e., sterilization) may not be required. Anesthetize healthy New Zealand
white rabbits weighing 1.5-2 kg with an intramuscular injection of a mixture of tiletamine hydrochloride and zolazepam hydrochloride (15 mg/kg) and xylazine hydrochloride (5 mg/kg). Verify anesthesia by monitoring the palpebral (blink) reflex or by ear pinch. NOTE: If an intraocular drug binds to ocular pigments, the degree of binding might induce modification of the drug's ocular pharmacokinetics. For example, the half-life of a pigment-binding drug in the vitreous and aqueous humors of
pigmented rabbits may be increased compared to that in albino rabbits14. In this case, the data obtained from pigmented rabbit eyes may be more similar and applicable to human eyes as human eyes have a different degree of pigmentation and albino rabbit eyes may not represent human counterparts. Thus, considering drug-pigment interaction, the use of pigmented or albino rabbits should be carefully considered and the results should be interpreted together with the strain (pigmentation)
of rabbit. However, albino rabbits are recommended for use in studies comparing pharmacokinetic properties as the drug-pigment interaction may be a confounding factor in the comparison. Apply a topical anesthetic with 1% proparacaine hydrochloride ophthalmic eye drops. Use vet ointment (2% hydroxypropyl methylcellulose) to prevent eye dryness until recovery from anesthesia. NOTE: Never leave the rabbit unattended while anesthetized. Dilate the pupil with one or two drops of phenylephrine hydrochloride and tropicamide. For surgical preparation before the intraocular injection, apply 5-10% povidone-iodine to the periocular skin with a cotton swab5. Place one drop of 5-10% povidone-iodine on the conjunctiva of the eye. Administer the intraocular drugs either intravitreally or intracamerally using aseptic techniques5. NOTE: The drug absorbed in systemic circulation may also penetrate the fellow eye. When a drug is administered to both eyes, the drug concentration in one eye may be affected by drug injected into the other eye. If it is confirmed that the effect of the contralateral injection can be neglected as the drug in systemic circulation cannot penetrate the other eye, the use of both eyes for intraocular injections can be considered, as it is economical and minimizes the number of animals sacrificed for the pharmacokinetic studies. For pharmacokinetic study on systemic concentration following intraocular injection, the use of only one eye is appropriate.
Observe the treated rabbits daily for one week, and once a week afterwards, for any sign of severe intraocular inflammation (conjunctival injection and hypopyon) until euthanasia. NOTE: Analgesics after injection are not required as intraocular injection using a 30 G needle is a minimally invasive procedure that is not accompanied by post-surgical pain. Do not return an animal that has undergone surgery to the company of the other animals until fully recovered. 2. Sample Preparation
3. ImmunoassayNOTE: Several analytical methodologies can be used for the measurement of protein concentration. Choose an appropriate quantitative method, depending on the detection range. Briefly, the selected ion monitoring mode of HPLC can detect picogram levels of molecule, whereas LC-MS/MS can detect nanogram and picogram levels of protein for profiling with MRM/PRM mode, respectively. The detection limit of ELISA is considered to be at the picogram level.
4. Pharmacokinetic Analysis MethodsNOTE: For PK analysis, one can use either compartmental or non-compartmental analysis. In compartmental analysis, disposition behavior of molecules can be explained by an equation (model). Thus, compartmental PK analysis can predict the concentration at any time t whereas the non-compartmental model cannot visualize or predict concentration-time profiles for other dosing regimens. However, fitting of compartmental models can be a complex and lengthy process. In contrast, assumptions are less restrictive in non-compartmental model. The non-compartmental method is simple and used commonly to calculate pharmacokinetic parameters such as half-life, clearance, and volume of distribution. We chose compartmental models for pharmacokinetic studies on anti-VEGF agents.
Representative ResultsThe procedure that is used to conduct intravitreal injections of a drug of interest in rabbit eyes with sterile techniques is shown in Figure 1. The treated eyes are enucleated at a scheduled time and stored at -80 °C. For the analysis, three compartments, the aqueous humor, the vitreous, and the retina/choroid, are separated from the frozen rabbit eyes, as demonstrated in Figure 2. Samples of the compartments are prepared for the ELISA. After incubation with a secondary antibody, optical density is measured in a 96-well plate, which contains known concentrations of the drug of interest for the standard curve and samples from the three compartments that were collected at multiple time points after the intravitreal injection (Figure 3). The concentration data that is calculated from the standard curve (Supplementary Figure 1) can be fitted to the pharmacokinetic model, and the pharmacokinetic parameters can be determined from the fitted line (Figure 4). In Figure 4, pharmacokinetics of intravitreally injected bevacizumab in vitrectomized and non-vitrectomized eyes were evaluated and compared. Compartmental PK analysis was performed, which provided the following equation to explain PK behavior. C(t) = C1exp(-k1t) + C'2 exp(-k2t) C (µg/mL): Concentration at any time t (hour) C1, C2: back-extrapolated intercepts of the distribution and elimination phase k1, k2: rate constants at the distribution and elimination phase As shown in Figure 4, the estimated concentration according to the fitted model matched quite well to the actual measured values. There were no significant differences in vitreous concentration of bevacizumab and pharmacokinetic parameters such as half-life between with and without vitrectomy. This experiment suggests that the role of the vitreous in the distribution and clearance of bevacizumab is insignificant. Figure 1:Procedure for Performing Intravitreal Injections of Intraocular Drugs in the Rabbit Eye Under Anesthesia. Aseptic techniques are used by applying 5% povidone-iodine drops and by skin preparation, and the intravitreal injection of the drug of interest is performed using a syringe fitted with a 30 G needle. Please click here to view a larger version of this figure. Figure 2:Separation of the Frozen Rabbit Eyeball into Three Compartments. Following scleral incision with a surgical blade and removal of the iris, the aqueous humor and vitreous can be separated. Subsequently, the retina/choroid can be separated carefully from the sclera, which is the white outer layer of the eyeball. Please click here to view a larger version of this figure. Figure 3:Enzyme-linked Immunosorbent Assay (ELISA) of the Intraocular Drug in the Samples Representing the Three Compartments. This figure shows a 96-well plate, which is used for the immunoassay. After incubation with a secondary antibody, a color change is noted in the wells. The optical density of the color depends on the concentration of the drug of interest. Please click here to view a larger version of this figure. Figure 4:Fitting the Observed Data to the Pharmacokinetic Model. In this experiment comparing the pharmacokinetics of bevacizumab in vitrectomized and non-vitrectomized eyes, the actual intravitreal concentration of bevacizumab is presented as dots. The fitted curves for the concentration data that are driven by the pharmacokinetic model, which are represented by two lines, are drawn and used for the calculation of pharmacokinetic parameters, such as the half-life of the drug. Error bars indicate 95% confidence intervals. Please click here to view a larger version of this figure. Supplementary Figure 1: The Standard Curve of the ELISA Used for Detection of Bevacizumab. This is obtained using the software capable of generating a four-parameter logistic curve fit. Please click here to download this file.
Table 1: Vitreous and Aqueous Humor Volume in Different Species 17,22-30 . DiscussionWith the increasing use of intraocular drugs, such as anti-vascular endothelial growth factor (VEGF) agents, for the treatment of diverse ocular diseases, knowledge of the tissue distribution and clearance of the drug after the intraocular injection is important. Understanding the pharmacokinetics of intraocular drugs is important for understanding the efficacy and safety of drugs, determining the optimal dosage of the drugs, and minimizing systemic or intraocular complications. However, detailed pharmacokinetic studies of intraocular drugs in humans are limited owing to safety issues, and an appropriate animal model is necessary. The rabbit system is a well-established model that is used to study the intracameral, intravitreal, and intraretinal pharmacokinetics of intraocular drugs16,21,31-33. Rabbits are easy to handle and use in experiments. In addition, animal studies that use rabbits are very economical as compared to those that use larger animals such as monkeys. For pharmacokinetic studies, this system allows the simultaneous sampling of the aqueous fluid, whole vitreous, retina, and serum from the same rabbit, and at different time points in different rabbits. Furthermore, rabbit eyes can be prepared to simulate treated human eyes, such as vitrectomized eyes, thus allowing pharmacokinetic studies under specific conditions15,19. With this model, researchers can analyze the effects of specific modulations or eye conditions to the pharmacokinetic properties of intraocular drugs. For example, previous studies of anti-VEGF agents in vitrectomized and non-vitrectomized eyes suggested that the removal of the vitreous by a vitrectomy does not affect the intraocular pharmacokinetics of anti-VEGF agents15,19. Our protocol consists of intraocular injection of the drug into the rabbit eyes, sample preparation, immunoassay, and pharmacokinetic analysis methods. Among these, the critical step within this protocol is sample preparation. Separation of the frozen eyeball into three compartments is not easy if the eyeball is thawed. Although the details of the separation are described in this protocol, quick separation of the three compartments while keeping the tissue frozen is the key step for successful partition. It is also possible to modify the sample preparation. For example, anterior chamber samples can be obtained by paracentesis of aqueous humor34,35. Vitreous sample can be also obtained by tapping35, which has not been used commonly owing to high failure rate and the possibility of tissue disruption. For the sample preparation of the retina, retinal tissues can be dissected into two layers with Ames solution36. The second important point is to determine the appropriate time-points at which to sacrifice rabbits and enucleate the eyeballs. We suggest 1 hr, and 1, 2, 5, 9, 14, and 30 days as sampling time points for large protein drugs, such as bevacizumab or ranibizumab. The half-life of an intravitreal drug depends primarily on its molecular weight. If the half-life of a drug of interest is expected to be shorter or longer than that of bevacizumab or ranibizumab, the sampling time points should be adjusted accordingly. Third, using one eye or both eyes of a rabbit is sometimes a matter of concern. Although the systemic absorption of an intraocular drug is usually minimal, intravitreal drugs can move to the contralateral eye and bias the result. Thus, the use of both eyes should be limited to performing a paired comparison of pharmacokinetics of two different drugs in the same animal or to comparing one single drug under two different ocular conditions (such as vitrectomized eyes). Fourth, we used ELISA, an immunologic assay, to measure the concentration of protein drugs. To measure the concentration of chemical drugs, high-performance liquid chromatography (HPLC) can be used. Multiple-reaction monitoring (MRM) using mass spectrometry is a recent quantitative method for analysis of proteins and peptides with high selectivity and sensitivity. If no reliable antibody-based assay is available for specific drugs, MRM can be a good alternative. One limitation of the method is that the differences between rabbit and human eyes could result in altered pharmacokinetics in the rabbit as compared to humans. In particular, humans have a vitreous cavity with a larger volume (4.5 ml) than that of rabbits (1.5 ml)21. In addition, because humans have a larger serum compartment than rabbits, the systemic exposure may be decreased. Furthermore, in humans, the vitreous becomes more liquefied and less homogenous with aging, which is associated with increased convective flow in the vitreous37. Therefore, liquefaction and convection in the vitreous have been suggested to be major pharmacokinetic differences between the rabbit and human38. These differences should be carefully considered when extrapolating the drug pharmacokinetic properties that are obtained from animal studies to humans. Another limitation is that this method measures the intraocular drug concentration in different animals. There might be differences in size, pharmacokinetic properties, and ocular and systemic conditions among rabbits in an experiment. To determine accurate pharmacokinetics, repeated longitudinal measurements in the same animal are preferable to using different animals. An alternative method is to use radioisotope-labeled drugs and measure the concentration with PET scans39. However, the PET scan usually has wide measurement deviations, and it is considered inappropriate for accurate measurement of low concentrations. A recent technology of intravital imaging could be a future research tool to overcome this limitation, for intraocular pharmacokinetic study40. As more drugs are developed for intravitreal injection, the need for intraocular pharmacokinetic studies will increase. In addition, various drug delivery systems such as fluocinolone acetonide intravitreal implant41-43 and dexamethasone intravitreal implant44,45, are being developed and the prediction of intraocular drug retention would be as important as intravitreal injection itself. Likewise, this protocol can be applied to other ocular delivery routes, such as topical, subconjunctival, and suprachoroidal46 administration. In addition, it can be applied to investigating the pharmacokinetics of long-term sustained intraocular drug-delivery systems, such as PLGA implants47, hydrogels, and microparticles. We believe that this protocol for the pharmacokinetic study of intraocular drugs using rabbit eyes will be useful to many researchers in ophthalmological fields. DisclosuresThe authors have no conflicts of interest to disclose. AcknowledgmentsWe would like to thank Ms. Ji Hyun Park and Ji Yeon Park for their technical assistance in the animal experiments. This work was supported by a grant from the Seoul National University Bundang Hospital Research Fund (grant number: Grant No. 14-2014-022) and from a grant (CCP-13-02-KIST) from the Convergence Commercialization Project of the National Research Council of Science and Technology, Seoul, Korea. References
Articles from Journal of Visualized Experiments : JoVE are provided here courtesy of MyJoVE Corporation Which of the following actions help prevent systemic absorption of a topical medication used in the eye?To decrease systemic absorption of topical medications, it is advisable for patients to use gentle punctal occlusion or eyelid closure for 2 minutes after drug instillation.
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