Name: use of rabbit eyes in pharmacokinetic studies of intraocular drugs
Uploaded: 2016-07-23T13:00:00
Description: Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

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 ...

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.

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			<td height="46" style="height:46px;width:213px;">Zoletil</td>
			<td style="width:143px;">Virbac Laboratories, Carros Cedex, France</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
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			<td height="46" style="height:46px;width:213px;">Xylazine hydrochloride&#160;</td>
			<td style="width:143px;">Fort Dodge Laboratories, Fort Dodge, IA</td>
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			<td height="46" style="height:46px;width:213px;">Proparacaine hydrochloride (Alcaine)</td>
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			<td height="40" style="height:40px;width:213px;">NUNC MICROWELL 96F&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; W/LID NUNCLON D SI&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td style="width:119px;">167008</td>
			<td style="width:173px;">96 well plate</td>
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			<td style="width:143px;">BOVOGEN</td>
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			<td height="40" style="height:40px;width:213px;">Phosphate Buffered Saline (PBS) pH7.4 (1X), 500mL</td>
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			<td height="60" style="height:60px;width:213px;">Sheep anti-Human IgG Secondary Antibody, HRP conjugate</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">PA1-28652</td>
			<td style="width:173px;"></td>
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			<td height="21" style="height:21px;width:213px;">Goat Anti-Human IgG Fc(HRP)</td>
			<td style="width:143px;">abcam</td>
			<td style="width:119px;">ab97225</td>
			<td style="width:173px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Goat anti-Human IgG, Fab&#39;2 Secondary Antibody, HRP conjugate</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">PA1-85183</td>
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			<td style="width:143px;">SIGMA</td>
			<td style="width:119px;">C3228-50ML</td>
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			<td height="21" style="height:21px;width:213px;">100 Scalpel Blades</td>
			<td style="width:143px;">nopa instruments</td>
			<td style="width:119px;">BLADE #15</td>
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			<td height="21" style="height:21px;width:213px;">100 Scalpel Blades</td>
			<td style="width:143px;">nopa instruments</td>
			<td style="width:119px;">BLADE #10</td>
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			<td height="40" style="height:40px;width:213px;">FEATHER SURGICAL BLADE STAINLESS STEEL</td>
			<td style="width:143px;">FEATHER</td>
			<td style="width:119px;">11</td>
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			<td height="40" style="height:40px;width:213px;">1-StepTM TMB-Blotting substrate solution, 250mL</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">34018</td>
			<td style="width:173px;"></td>
		</tr>
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			<td height="40" style="height:40px;width:213px;">Stable Peroxide Substrate Buffer (10X), 100mL</td>
			<td style="width:143px;">Thermo SCIENTIFIC</td>
			<td style="width:119px;">34062</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Softmax Pro</td>
			<td style="width:143px;">Molecular Devices</td>
			<td style="width:119px;">v.5.4.1</td>
			<td style="width:173px;">software for generating standard curve</td>
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			<td height="63" style="height:63px;width:213px;">SAAM II&#160;</td>
			<td style="width:143px;">Saam Institute, Seattle, WA</td>
			<td style="width:119px;"></td>
			<td style="width:173px;">software for pharmacokinetic modeling</td>
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			<td height="63" style="height:63px;">Phoenix WinNonlin</td>
			<td style="width:143px;">Pharsight, Cary, NC</td>
			<td style="width:119px;">v. 6.3</td>
			<td style="width:173px;">software for pharmacokinetic modeling</td>
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			<td height="21" style="height:21px;width:213px;">Avastin (bevacizumab)</td>
			<td style="width:143px;">Genentech</td>
			<td style="width:119px;"></td>
			<td style="width:173px;"></td>
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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 &#176;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 incuba...

Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

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. ...

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