Name: Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs (Video) | JoVE Journal
Uploaded: 2016-07-23T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Learn pharmacokinetic profiling of intraocular drugs using rabbit eyes to study drug distribution in ocular compartments. This method enables researchers to optimize ocular drug delivery strategies.

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5 minutes

We 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.

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.
1. Intraocular Injection of the Drug into Rabbit Eyes
<ol>
	<li>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&#39;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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Dilate the pupil with one or two drops of phenylephrine hydrochloride and tropicamide.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>For intravitreal injections, inject the drug intravitreally 1 mm behind the surgical limbus in the superotemporal quadrant using a 30 G needle and either commercial 1 ml syringes, insulin syringes, or glass syringes perpendicularly to the scleral surface5. 
		NOTE: The volume of drug used for intracameral and intravitreal injection varies depending on the drugs under investigation. For pharmacokinetic experiments on anti-VEGF agents using rabbit eyes, previous studies used 0.025, 0.05 (most common), or 0.1 ml of bevacizumab or ranibizumab for the intracameral or intravitreal injection. In our previous studies on intraocular pharmacokinetics of anti-vascular endothelial growth factor, 0.05 ml of bevacizumab15, 0.025 ml of ranibizumab, or 0.03 ml of VEGF-Trap16 were used. The largest safe volume without paracentesis is believed to be 0.1 ml, although there has been little evidence to support this17. If excessive volume is injected either intracamerally or intravitreally, intraocular pressure is greatly increased. In addition to optic nerve damage directly caused by increased intraocular pressure (IIOP), namely glaucomatous optic nerve damage, in extreme cases the IIOP leads to impaired ocular perfusion and central retinal artery occlusion, which is analogous to stroke in the brain.</li>
		<li>For intracameral injections, inject the drug into the anterior chamber by inserting a 30 G needle through the corneoscleral limbus with the bevel up and advancing it parallel to the iris plane to minimize the risk of trauma to the iris or lens. 
		NOTE: Depending on the drug formulation, needles larger than 30 G can be used. The conditions requiring larger-gauge needle include drugs containing microspheres, large-protein drugs, and high viscosity formulations.</li>
		<li>After injection, compress the injection site with a sterile cotton-tip applicator to promote wound healing. 
		NOTE: Normally, 30 sec&#160;is enough for wound healing, when a 30 G needle is used for intraocular injection. However, if a larger gauge needle is used for intraocular injection, longer time would be appropriate for wound sealing to minimize the leakage from the sclerotomy wound. To check for sclerotomy wound leakage is important to ensure that the amount of intraocular drug used immediately after the intraocular injection is the same as the injected amount, especially if a larger gauge needle is used.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Sample Preparation
<ol>
	<li>For enucleation, euthanize the rabbits at various time points (e.g., 1 hr or 1, 2, 5, 9, 14, or 30 days) after the intraocular drug injection. 
	NOTE: These time points depend on the drug of interest and the known pharmacokinetic profiles. For each time point, use at least two eyeballs13. For reliable data quality, sampling time should be chosen carefully, at least four time-points with balanced sampling for at least a time-span of two half-lives of the drug13. We believe that 6-8 time-points with balanced sampling may be appropriate. For small molecules (&#8804;1,000 Da)18, in particular, more than 1 time-point during the first 24 hr is crucial. Accordingly, for macromolecules (&#62;1,000 Da)18, a sampling period such as 1 hr and 1, 2, 5, 9, 14, and 30 days19 can be a good option. For small molecules (&#8804;1,000 Da), 1, 2, 4, and 8 hr and 1, 3, and 7 days can be one option20. Depending on the molecular weight, the sampling period can be further modified.
	<ol>
		<li>For euthanasia, administer 10 ml of 15% intravenous KCl rapidly after anesthetizing rabbits with an intramuscular injection of a mixture of tiletamine hydrochloride, zolazepam hydrochloride (15 mg/kg), and xylazine hydrochloride (5 mg/kg).</li>
	</ol>
	</li>
	<li>Open the palpebral fissure with an eyelid retractor. Make a 360&#176; conjunctival incision 2-3 mm posterior to the limbus and extend it posteriorly by dissecting the conjunctiva and Tenon&#39;s capsule from the globe.</li>
	<li>Cut the extraocular muscles close to their insertion into the globe. Clamp the optic nerve with curved forceps and then cut the nerve between the forceps and the globe. Remove the eyeball itself while leaving the surrounding tissues intact. After enucleation, immediately freeze the eyeballs and store them at -80 &#176;C. 
	NOTE: There may be two options for immediate freezing. If the drug concentration is measured at a very early phase (i.e. less than 1 hr), liquid nitrogen may be more appropriate to ensure better timely freezing of the eyeball. However, for subsequent periods, ice can be used to cool the eyeball immediately, and then deep freezer can be used to store the eyeballs at -80 &#176;C.</li>
	<li>After obtaining the eyeballs for all of the time points, separate the frozen eyeballs into three compartments, the vitreous, the aqueous humor, and the retina/choroid. Separate these compartments before defrosting. 
	NOTE: The most important point for the separation into three compartments is the speed of the procedure. The eyeball&#160;should be separated very quickly before defrosting and the procedure can be performed on the ice to delay thawing.
	<ol>
		<li>To open the globe, make an incision at the corneal limbus (300&#176; or greater) with a scalpel blade. Obtain the frozen compartment in front of the iris, the aqueous humor.</li>
		<li>Remove the frozen iris and lens by pulling and excoriating using tissue forceps. When the vitreous becomes accessible, obtain the frozen vitreous by separating it from the remaining tissues (retina/choroid/sclera).</li>
		<li>Using a No. 15 scalpel blade, separate the retina/choroid tissue from the underlying sclera.</li>
	</ol>
	</li>
	<li>For immunoassays, defrost the aqueous humor samples, and measure the volume of each sample. Measure the weight of the frozen samples by subtracting the weight of an empty tube from that of the tube containing the frozen sample. 
	NOTE: Because the specific gravity of the frozen samples is approximately 1, the weight of each sample can be used to calculate the volume of the sample.</li>
	<li>Weigh the vitreous samples, defrost the samples, and solubilize them in 1.0 ml of phosphate-buffered saline containing 1% bovine serum albumin on a rotator overnight at 4 &#176;C. Then, centrifuge the samples at 387 x g for 10 min21.</li>
	<li>Weigh the frozen retina/choroid samples for homogenization. Add protein extraction reagent with a ratio of tissue to reagent of 1:10 (1 g of tissue/10 ml of reagent). Homogenize the tissue using a pre-chilled microhomogenizer. Centrifuge the lysed sample for 10 min at 12,000-20,000 x g and transfer the supernatant to a chilled EPP tube.</li>
</ol>
3. Immunoassay
NOTE: 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.
<ol>
	<li>For the indirect enzyme-linked immunosorbent assays (ELISAs)&#160;to measure concentrations of anti-VEGF agents, use ELISA kits in 96-well plates to detect the drug of interest and generate a standard curve of known drug concentrations.
	<ol>
		<li>Dilute the vitreous, the aqueous humor, and the retina/choroid samples with 0.1% bovine serum albumin in 1x phosphate-buffered saline (PBS) to concentrations that are within the linear range, and use these for the assay.</li>
	</ol>
	</li>
	<li>Divide the samples into aliquots on the plate at 100 &#181;l/well. Incubate overnight at 4 &#176;C and then wash the plate three times using 200 &#181;l of the washing solution of 0.05% Tween20 in 1x PBS. 
	NOTE: The anti-VEGF present in the sample acts as the primary antibody for ELISA; therefore, additional use of a primary antibody is not necessary.</li>
	<li>Dilute the secondary antibody to 1:20,000 in 0.1% BSA in 1x PBS, and add 100 &#181;l of the diluted-solution per well. After incubating the plate with a diluted secondary antibody overnight at 4 &#176;C, measure the optical density at 450 nm wavelength. Subtract the average zero standard optical density from the average of duplicate readings for each standard and sample.</li>
	<li>Create a standard curve based on the relative light signal from solutions of the drug with known concentrations by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve fit, such as SoftMax Pro. For creating a standard curve, the 4-PL curve fit can be obtained by clicking on [4-parameter] in [Standard Curve] - [Fit].</li>
	<li>Calculate the drug concentration in the samples from the standard curve. 
	NOTE: The limits of detection (LOD) of anti-VEGF drugs were investigated in our experiment. The LOD of bevacizumab was 0.024-3.125 ng/ml and that of aflibercept was 0.039-10 ng/ml.</li>
</ol>
4. Pharmacokinetic Analysis Methods
NOTE: 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.
<ol>
	<li>Analyze the drug concentration data with the compartmental models using modeling software such as Phoenix WinNonlin software.
	<ol>
		<li>Click [PK model] in [WNL5 classic modeling] and map the observation time, administered dose, and drug concentration in the [Setup] menu.</li>
		<li>Select compartmental model (e.g., number of compartments) in the [model selection] tab, and click the [Execution] button to calculate model parameters.</li>
	</ol>
	</li>
	<li>After the analyses, select the final compartment model that best describes the drug concentration data based on the following criteria: (1) Akaike Information Criterion, (2) precision of parameter estimates (e.g., standard error), and (3) graphical analysis (e.g., goodness of fit plots).</li>
	<li>Calculate pharmacokinetic parameters of interest, such as half-life (T1/2) and area under the time-concentration curve (AUC), from the model parameters and equations driven by the compartmental model.</li>
</ol>

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<li>Scholes, G. N., O'Brien, W. J., Abrams, G. W., Kubicek, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clearance+of+triamcinolone+from+vitreous%5BTitle%5D)+AND+%22Arch+Ophthalmol%22%5BJournal%5D)">Clearance of triamcinolone from vitreous.</a> Arch Ophthalmol. 103, 1567-1569 (1985).</li>
<li>Stastna, M., Behrens, A., McDonnell, P. J., Van Eyk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+protein+composition+of+rabbit+aqueous+humor+following+two+different+cataract+surgery+incision+procedures+using+2-DE+and+LC-MS%2FMS%5BTitle%5D)+AND+%22Proteome+Sci%22%5BJournal%5D)">Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS.</a> Proteome Sci. 9, 8(2011).</li>
<li>Sinapis, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacokinetics+of+intravitreal+bevacizumab+%28Avastin%28R%29%29+in+rabbits%5BTitle%5D)+AND+%22Clin+Ophthalmol%22%5BJournal%5D)">Pharmacokinetics of intravitreal bevacizumab (Avastin(R)) in rabbits.</a> Clin Ophthalmol. 5, 697-704 (2011).</li>
<li>Gaudreault, J., Fei, D., Rusit, J., Suboc, P., Shiu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+pharmacokinetics+of+Ranibizumab+%28rhuFabV2%29+after+a+single+intravitreal+administration%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration.</a> Invest Ophthalmol Vis Sci. 46, 726-733 (2005).</li>
<li>Maurice, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review%3A+practical+issues+in+intravitreal+drug+delivery%5BTitle%5D)+AND+%22J+Ocul+Pharmacol+Ther%22%5BJournal%5D)">Review: practical issues in intravitreal drug delivery.</a> J Ocul Pharmacol Ther. 17, 393-401 (2001).</li>
<li>Laude, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravitreal+therapy+for+neovascular+age-related+macular+degeneration+and+inter-individual+variations+in+vitreous+pharmacokinetics%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Intravitreal therapy for neovascular age-related macular degeneration and inter-individual variations in vitreous pharmacokinetics.</a> Prog Retin Eye Res. 29, 466-475 (2010).</li>
<li>Christoforidis, J. B., Carlton, M. M., Knopp, M. V., Hinkle, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PET%2FCT+imaging+of+I-124-radiolabeled+bevacizumab+and+ranibizumab+after+intravitreal+injection+in+a+rabbit+model%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">PET/CT imaging of I-124-radiolabeled bevacizumab and ranibizumab after intravitreal injection in a rabbit model.</a> Invest Ophthalmol Vis Sci. 52, 5899-5903 (2011).</li>
<li>Sangwan, V. S., Pearson, P. A., Paul, H., Comstock, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+the+Fluocinolone+Acetonide+Intravitreal+Implant+for+the+Treatment+of+Noninfectious+Posterior+Uveitis%3A+3-Year+Results+of+a+Randomized+Clinical+Trial+in+a+Predominantly+Asian+Population%5BTitle%5D)+AND+%22Ophthalmol+Ther%22%5BJournal%5D)">Use of the Fluocinolone Acetonide Intravitreal Implant for the Treatment of Noninfectious Posterior Uveitis: 3-Year Results of a Randomized Clinical Trial in a Predominantly Asian Population.</a> Ophthalmol Ther. 4, 1-19 (2015).</li>
<li>Bajwa, A., Aziz, K., Foster, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+efficacy+of+fluocinolone+acetonide+intravitreal+implant+%280.59+mg%29+in+birdshot+retinochoroidopathy%5BTitle%5D)+AND+%22Retina%22%5BJournal%5D)">Safety and efficacy of fluocinolone acetonide intravitreal implant (0.59 mg) in birdshot retinochoroidopathy.</a> Retina. 34, 2259-2268 (2014).</li>
<li>Sanford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluocinolone+acetonide+intravitreal+implant+%28Iluvien%28R%29%29%3A+in+diabetic+macular+oedema%5BTitle%5D)+AND+%22Drugs%22%5BJournal%5D)">Fluocinolone acetonide intravitreal implant (Iluvien(R)): in diabetic macular oedema.</a> Drugs. 73, 187-193 (2013).</li>
<li>Haller, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dexamethasone+intravitreal+implant+in+patients+with+macular+edema+related+to+branch+or+central+retinal+vein+occlusion+twelve-month+study+results%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Dexamethasone intravitreal implant in patients with macular edema related to branch or central retinal vein occlusion twelve-month study results.</a> Ophthalmology. 118, 2453-2460 (2011).</li>
<li>Boyer, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-year%2C+randomized%2C+sham-controlled+trial+of+dexamethasone+intravitreal+implant+in+patients+with+diabetic+macular+edema%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Three-year, randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with diabetic macular edema.</a> Ophthalmology. 121, 1904-1914 (2014).</li>
<li>Patel, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+administration+into+the+suprachoroidal+space+using+a+microneedle+for+drug+delivery+to+the+posterior+segment+of+the+eye%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye.</a> Invest Ophthalmol Vis Sci. 53, 4433-4441 (2012).</li>
<li>Makadia, H. K., Siegel, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poly+Lactic-co-Glycolic+Acid+%28PLGA%29+as+Biodegradable+Controlled+Drug+Delivery+Carrier%5BTitle%5D)+AND+%22Polymers+%28Basel%29%22%5BJournal%5D)">Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier.</a> Polymers (Basel). 3, 1377-1397 (2011).</li>
</ol>

The authors have no conflicts of interest to disclose.

With 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 ...

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 &#181;l) 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.

<table><tbody><tr><td>Zoletil</td><td>Virbac Laboratories, Carros Cedex, France</td><td></td><td></td></tr><tr><td>Xylazine hydrochloride&amp;nbsp;</td><td>Fort Dodge Laboratories, Fort Dodge, IA</td><td></td><td></td></tr><tr><td>Proparacaine hydrochloride (Alcaine)</td><td>Alcon laboratories, Fort Worth, TX</td><td></td><td></td></tr><tr><td>Phenylephrine hydrochloride and tropicamide</td><td>Santen Pharmaceutical, Co., Osaka, Japan</td><td></td><td></td></tr><tr><td>Recombinant Human VEGF 165</td><td>R&amp;amp;D systems</td><td>293-VE-050</td><td></td></tr><tr><td>Carbobate-Bicarbonate buffer</td><td>SIGMA</td><td>C3041-50CAP</td><td></td></tr><tr><td>Nunc Microwell 96F w/lid 
Nunclon D Si</td><td>Thermo SCIENTIFIC</td><td>167008</td><td>96 well plate</td></tr><tr><td>Bovine Serum Albumin (BSA) 25 g(Net)</td><td>BOVOGEN</td><td>BSA025</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4 (1x), 500 ml</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Sheep anti-Human IgG Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-28652</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc(HRP)</td><td>abcam</td><td>ab97225</td><td></td></tr><tr><td>Goat anti-Human IgG, Fab&#039;2 Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-85183</td><td></td></tr><tr><td>CelLytic MT&amp;nbsp; Cell Lysis Reagent</td><td>SIGMA</td><td>C3228-50ML</td><td>lysis buffer</td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #15</td><td></td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #10</td><td></td></tr><tr><td>Feather surgical blade stainless steel</td><td>FEATHER</td><td>11</td><td></td></tr><tr><td>1-StepTM TMB-Blotting substrate solution, 250 ml</td><td>Thermo SCIENTIFIC</td><td>34018</td><td></td></tr><tr><td>Stable Peroxide Substrate Buffer (10x), 100 ml</td><td>Thermo SCIENTIFIC</td><td>34062</td><td></td></tr><tr><td>Softmax Pro</td><td>Molecular Devices</td><td>v.5.4.1</td><td>software for generating standard curve</td></tr><tr><td>SAAM II&amp;nbsp;</td><td>Saam Institute, Seattle, WA</td><td></td><td>software for pharmacokinetic modeling</td></tr><tr><td>Phoenix WinNonlin</td><td>Pharsight, Cary, NC</td><td>v. 6.3</td><td>software for pharmacokinetic modeling</td></tr><tr><td>Avastin (bevacizumab)</td><td>Genentech</td><td></td><td></td></tr></tbody></table>

use of rabbit eyes in pharmacokinetic studies of intraocular drugs

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

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.

The 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 ...

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

The overall goal of this experiment is to investigate the pharmacokinetic properties of intraocular drugs using rabbit eyes. This method can help answer key questions in the ocular pharmacology field, such as the pharmacokinetics of intravitreally injected drugs. The main advantage of this technique is the rabbit eyeballs are relatively large, and thus intravitreal injection techniques and intraocular distribution of injected drugs can be comparable to humans.In addition, rabbits are easier to handle and less expensive than primates. The implications of this technique stands toward intraocular drug delivery, because use of drug delivery greatly affect the drug concentration and duration of action in ocular compartment. Thus, drug concentrations over time should be investigated in each compartment.Demonstrating the procedure will be Hye Kyoung Hong, a post-doc from my laboratory. After anesthetizing a healthy New Zealand white rabbit, according to the text protocol, apply a topical anesthetic eyedrop, then apply wetting ointment or gel onto the cornea to prevent eye dryness until recovery from anesthesia. To dilate the pupil, apply one or two drops of phenylephrine hydrochloride, and trapicamide.Use a cotton swab to apply 10 percent povidone iodine to the periocular skin. Then place one drop of five percent povidone iodine on the conjunctiva of the eye. Next, using a 30 gauge needle attached to a syringe, inject the drug of interest intravitreally, one millimeter behind the surgical limbus in the supratemporal quadrant, perpendicularly to the scleral surface.Minimizing leakage from the sclera is important, as leakage can be a source of bias. To prevent leakage from the vitreous cavity, a cotton tip should be applied following the needle removal, and compression should be applied for at least 30 seconds. After allowing enough time for wound healing, observe the treated rabbits daily for one week, and once a week afterwards for any sign of severe intraocular inflammation.After euthanizing the rabbits according to the text protocol, use an eyelid retractor to open the palpebral fissure. Two to three millimeters posterior to the limbus make a 360 degree conjunctival incision and extended posteriorally by dissecting the conjunctiva and tenon's capsule from the globe. Cut the extraocular muscles close to their insertion into the globe.Then, with curved forceps, clamp the optic nerve and cut the nerve between the forceps and the globe. Remove the eyeball itself while leaving the surrounding tissues intact. Then, using liquid nitrogen, immediately freeze the eyeballs and store them at 80 degrees Celsius.After isolating the eyeballs and freezing them for all of the time points, before defrosting, quickly separate the frozen eyeballs into three compartments. The vitreous, the aqueous humor, and the retina choroid. Separation of the frozen eyeball into three compartments is not an easy task if the eyeball is thawed.Quick separation of the three compartments while keeping the tissue frozen is the key step for success for partition. Next, to open the globe, use a scalpel blade to make an incision in the corneal limbus, and incise and separate the anterior segment from the posterior segment, then isolate the aqueous humor, which is the frozen compartment in front of the iris. With tissue forceps, pull and excoriate the frozen iris and lens to remove the tissues.Then when the vitreous becomes accessible, separate it from the remaining retina, choroid and sclera. Now, using a number 15 scalpel blade, separate the retinochoroid tissue from the underlying sclera. For immunoassays, defrost the aqueous humor samples and measure the volume of each sample.Determine the weight of the frozen samples by subtracting the weight of an empty tube from that of the tube containing the frozen sample. After weighing the vitreous samples, use PBS with one percent BSA to defrost and solubilize the tissue. Then place on a rotator at four degrees Celsius overnight.Then, centrifuge the tissue at 387 times G for 10 minutes. To homogenize the retinochoroid samples after weighing the frozen tissue, add protein extraction reagent at a one to 10 ratio of tissue to reagent. Then using a pre-chilled tissue microhomogenizer, homogenize the tissue.Centrifuge the lysed sample for 10 minutes at 12, 000 to 20, 000 times G, and transfer the supernatant to a chilled EPP tube. To carry out an ELISA, use 0.1 percent BSA in one XPBS to dilute the vitreous, aqueous humor, and retinochoroid samples to concentrations that are within the limit of detection, and use these for the assay. Aliquot 100 microliters per well of the samples and known concentrations of the drug of interest to the 96 well plate to construct a standard curve.Then incubate the plate overnight at four degrees celsius, before using 200 microliters of washing solution to wash the plate three times. Dilute the secondary antibody one to 20, 000 in PBS with 0.1 percent BSA, and add 100 microliters to each well of the assay plate. Following the incubation of the plate at four degrees Celsius overnight, measure the OD 450.After creating a standard curve and calculating drug concentrations in the samples according to the text protocol, analyze the drug concentration data with compartmental models using modeling software such as Phoenix Winnonlin software. In WNL5 classic modeling, click PK model, and in the setup menu, map the observation time, administered dose, and drug concentration. Select the final compartment model and calculate pharmecokinetic parameters of interest according to the text protocol.Following intravitreal injections of a drug of interest, and preparation of tissue samples as demonstrated in this video, ELISA was carried out to determine the concentration of the drug in the vitreous, the aqueous humor, and the retinochoroid. As shown here, the optical density of the color correlates with the concentration of the drug of interest. The concentration data that is calculated from the standard curve can be fitted to the pharmecokinetic model, and the pharmecokinetic parameters can be determined from the fitted line.In this experiment, the pharmecokinetics of intravitreally injected Bevacizumab, in vitrectomized, and to non-vitrectomized eyes for multiple timepoints were evaluated and compared using the following equation. As shown here, the estimated concentration according to the fitted model, matched quite well to the actual measured values. There were no significant differences in vitreous concentration or half-life of Bevacizumab, suggesting that the role of vitreous in the distribution and clearance of Bevacizumab is insignificant.Once mastered, the critical eyeball separation technique can be done in 10 minutes if it is performed properly. While attempting this procedure, it is important to remember that the time points for sacrificing rabbits and inoculating their eyeballs should be individualized to the drug of interest, as pharmacokinetic parameters such as molecular weight can vary widely. After its development, this technique paved the way for researchers in the field of ocular pharmacology to explore intraocular pharmacokinetics in a rabbit eye.After watching this video, you should have a good understanding of how to perform an intraocular pharmacokinetic study using rabbit eyes. Don't forget that the differences between rabbit and human eyes should be carefully considered when extrapolating the draw pharmacokinetic properties that are obtained from animal studies to humans.

use-of-rabbit-eyes-in-pharmacokinetic-studies-of-intraocular-drugs

Research

JoVE Journal

Medicine

In this video, we show a procedure to induce aqueous-deficient dry eye disease in a rabbit model by injecting the palpebral portion of the superior lacrimal gland with Concanavalin A. This model is useful for pharmacokinetic and biodistribution assays of drugs for treating dry eye disease.

Performing Injection into Palpebral Portion of the Superior Lacrimal Gland: A Technique for Inducing Dry Eye Disease in Rabbit Model

This video describes the procedure to induce dry eye disease in a rabbit model through an injection of Concanavalin A into the orbital lobe of the superior lacrimal gland. The developed model is useful to study the effectiveness of various drugs for treating dry eye disease.

Performing Injection into Orbital Lobe of Superior Lacrimal Gland: A Technique for Inducing Dry Eye Disease in Rabbit Model

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes a technique for intracameral injection in rats using a central corneal incision and a long tunnel into the anterior chamber. This injection method minimizes the risk of inducing inadvertent tissue damage and thereby improves precision and reproducibility.

Intracameral Injection in Rats with Low Risk of Adverse Effects

This paper presents detailed instructions to perform optic nerve crush in a rabbit model using standard ophthalmic surgical techniques.

A Surgical Approach for Optic Nerve Crush in a Rabbit Model

This study presents a methodology for the delivery of therapeutics into the retina and optic nerves of the adult rat. Additionally, a unique tissue retrieval method is introduced for a top-down en bloc collection of the optic nerve and retina in an adult rat.

Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

Learn pharmacokinetic profiling of intraocular drugs using rabbit eyes to study drug distribution in ocular compartments. This method enables researchers to optimize ocular drug delivery strategies.

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

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