Pharmacokinetics of Intravitreal Ranibizumab (Lucentis)

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Pharmacokinetics of Intravitreal Ranibizumab (Lucentis) Sophie J. Bakri, MD,1 Melissa R. Snyder, PhD,2 Joel M. Reid, PhD,3 Jose S. Pulido, MD,1 Mohamed K. Ezzat, BS,1 Ravinder J. Singh, PhD2 Purpose: To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare it with that of 1.25 mg of intravitreal bevacizumab (Avastin), using the same rabbit model. Design: Experimental animal study. Participants: Twenty-eight Dutch-belted rabbits. Methods: One eye of each of 20 rabbits was injected with 0.5 mg of intravitreal ranibizumab. Both eyes of each of 4 rabbits were enucleated at days 1, 3, 8, 15, and 29. Ranibizumab concentrations were measured in aqueous fluid, whole vitreous, and serum. A further 8 rabbits were used to measure serum and fellow ranibizumab at additional time points of 3 and 8 hours. Main Outcome Measures: Ranibizumab concentrations in the aqueous, vitreous, and serum. Results: Although vitreous concentrations of ranibizumab declined in a monoexponential fashion with a half-life of 2.88 days, concentrations of ⬎0.1 ␮g/ml ranibizumab were maintained in the vitreous humor for 29 days. Ranibizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of 17.9 ␮g/ml, 3 days after drug administration. Elimination of ranibizumab from the aqueous humor paralleled that found in the vitreous humor, with a half-life value of 2.84 days. No ranibizumab was detected in the serum or the fellow eye. Conclusion: In the rabbit, the vitreous half-life of 0.5-mg intravitreal ranibizumab is 2.88 days, shorter than the half-life of 1.25-mg intravitreal bevacizumab of 4.32 days. No ranibizumab was detected in the serum or the fellow uninjected eye; whereas small amounts of intravitreal bevacizumab have been detected in the serum and fellow uninjected eye. Ophthalmology 2007;114:2179 –2182 © 2007 by the American Academy of Ophthalmology.

Ranibizumab (Lucentis, Genentech Inc., San Francisco, CA) is a recombinant humanized monoclonal immunoglobulin G1 (IgG1) ␬-isotype antibody fragment that inhibits human vascular endothelial growth factor (VEGF). It is approved by the United States Food and Drug Administration for intravitreal injection for choroidal neovascularization due to age-related macular degeneration.1,2 Monthly injection of the approved 0.5-mg dose prevented vision loss and increased mean visual acuity at 2 years. Bevacizumab (Avastin, Genentech) is a recombinant humanized monoclonal IgG1 antibody that inhibits human VEGF. It is approved by the U.S. Food and Drug Administration for intravenous use for metastatic colorectal cancer but is used off-label intravitreally to treat VEGF-mediated diseases such as choroidal neovascularization,3–5 central retinal vein occlusion,6,7 and proliferative diabetic retinopathy.8 –10 Because intravitreal bevacizumab is an off-label treatment, there are no specific recommendations as to the freOriginally received: June 19, 2007. Final revision: August 24, 2007. Accepted: September 13, 2007. Manuscript no. 2007-820. 1 Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota. 2 Department of Biochemistry, Mayo Clinic, Rochester, Minnesota. 3 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota. © 2007 by the American Academy of Ophthalmology Published by Elsevier Inc.

quency of its dosing. We have recently described the pharmacokinetics of intravitreal bevacizumab in a rabbit model11 and have shown that the half-life of intravitreal bevacizumab is 4.32 days in the vitreous cavity. The purpose of this study is to use the same rabbit model to describe the pharmacokinetics of intravitreal ranibizumab, to enable a direct comparison of the pharmacokinetics of intravitreal bevacizumab and ranibizumab.

Materials and Methods Approval was obtained from the Institutional Animal Care and Use Committee at the Mayo Clinic, and the procedures adhered to the guidelines from the Association for Research in Vision and Ophthalmology for animal use in research. Twenty Dutch-belted male rabbits weighing 1.7 to 2 kg (Harlan Laboratories, Indianapolis,

Presented in part at: Bascom Palmer Eye Institute Angiogenesis Meeting, February 2007, Key Biscayne, Florida. Supported by an unrestricted grant from Research to Prevent Blindness, New York, New York. No author has any proprietary interest in any of the products mentioned in the article. No conflicting interest exists for any author. Correspondence to Sophie J. Bakri, MD, Department of Ophthalmology, Mayo Clinic, Rochester, MN 55905. E-mail: [email protected]. ISSN 0161-6420/07/$–see front matter doi:10.1016/j.ophtha.2007.09.012

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Ranibizumab Assay Ranibizumab concentrations were measured using an immunoassay technique. The 165–amino acid variant of human recombinant VEGF (R&D Systems, Minneapolis, MN) was immobilized on Microlite 2 (Thermo Labsystems, Franklin, MA) high-binding plates designed for luminescent detection. The VEGF was diluted to a concentration of 1.0 ␮g/ml in a 50-mmol/l carbonate buffer, pH 9, then aliquoted onto the Microlite plates at 100 ␮l/well. After an overnight incubation at 4° C, the plates were washed 3 times with ⫻1 PBS on an EL50 AutoStrip Washer (Bio-Tek Instruments, Inc., Winooski, VT) and then blocked for 4 hours at 4° C with 1% bovine serum albumin in ⫻1 PBS. After 3 washes with ⫻1 PBS on the AutoStrip Washer, the plates were stored dry at 4° C. Samples to be assayed were diluted so as to be within the linear range of the assay. Samples were diluted in StabilCoat reagent (Surmodics, Inc., Eden Prairie, MN), aliquoted onto a VEGF plate at 100 ␮l/well, and then incubated for 2 hours at room temperature with agitation. For each individual assay, a standard curve was included using ranibizumab of known concentrations ranging from 12 ng/ml to 0.375 ng/ml. After the initial incubation, the plates were washed 3 times with 0.05% Tween 20 in 1⫻ PBS on the AutoStrip Washer. The bound ranibizumab was detected with a goat antihuman IgG/F(ab=)2 antibody labeled with horseradish peroxidase (Pierce Biotechnology Inc., Rockford, IL). The labeled anti– humanized monoclonal IgG/F(ab=)2 was diluted 1:20 000 in StabilCoat reagent. The diluted secondary antibody was aliquoted onto the VEGF plate at 100 ␮l/well and incubated for 45 minutes at room temperature with agitation. After this incubation, the plate was washed 3 times with 0.05% Tween 20 in 1⫻ PBS on the AutoStrip Washer. The chemiluminescent signal was triggered using the luminol-based SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce Biotechnology) according to the manufacturer’s instructions. The chemiluminescent signal was detected on a PHERAstar Microplate Reader (BMG Labtech, Durham, NC) and acquired over a time frame of 0.1 seconds/well.

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The concentration of ranibizumab in the unknown samples was calculated from the standard curve, which was constructed using the relative light signal from solutions of ranibizumab with known concentrations. The equation of the line from the standard curve was calculated by linear regression. This line equation was used to calculate the concentration of ranibizumab in the aqueous, vitreous, and serum from the injected rabbits.

Pharmacokinetic Methods Ranibizumab concentration–time data were fit by standard noncompartmental analysis to determine half-life, AUC (0 –⬁), and vitreous clearance using WinNonlin Pro (version 4.1, Pharsight, Mountain View, CA). The terminal elimination rate constant was determined by least-squares regression of the ln(serum concentration)–time data for the last 4 time points.

Results Data were obtained from the 48 eyes of 28 rabbits. No adverse events were noted, and there were no signs of ocular inflammation. The sensitivity of the assay was 0.375 ng/ml. The change in concentration over time for ranibizumab in vitreous and aqueous humor, both from the injected eye, is illustrated in Figure 1. A peak concentration of 162 ␮g/ml was achieved in the vitreous humor 1 day after intravitreal injection of 0.5 mg of ranibizumab. Vitreous concentrations of ranibizumab declined in a monoexponential fashion with a half-life of 2.88 days. Concentrations of ⬎0.1 ␮g/ml ranibizumab were maintained in the vitreous humor for 29 days. Ranibizumab concentrations in the aqueous humor of the injected eye reached a peak of 17.9 ␮g/ml, 3 days after drug administration. Elimination of ranibizumab from the aqueous humor paralleled that found in the vitreous humor, with a half-life of 2.84 days. Mean residence times (time to clear ⬎69% of the drug) in vitreous and aqueous were 4 and 6.8 days, respectively. To ensure that ranibizumab was not cleared from the serum during the 24-hour period before the first sample was drawn for the pharmacokinetic study, the experiment was repeated using 4 rabbits at each of 2 further time points at 3 and 8 hours. No ranibizumab was detected in the serum of these rabbits.

Ranibizumab concentration (µg/ml)

IN) were anesthetized with 35 mg/kg of intramuscular ketamine hydrochloride (Fort Dodge Inc., Fort Dodge, IN), 5 mg/kg of intramuscular xylazine hydrochloride (Phoenix Scientific Inc., St. Joseph, MO), and 1% proparacaine hydrochloride ophthalmic drops (Allergan America, Hormigueros, Puerto Rico) topically on the eye. Povidone–iodine 5% was placed on the conjunctiva of 20 right eyes. These right eyes were injected intravitreally 1 mm behind the surgical limbus in the superotemporal quadrant with 0.5 mg (0.05 cm3) of ranibizumab, using a 30-gauge needle. The 20 left eyes were controls and received no intravitreal injections. Eyes were monitored weekly for signs of inflammation. Four rabbits were sacrificed at each of the time points days 1, 3, 8, 15, and 29 by intravenous pentobarbital overdose (Beuthanasia-D Special, Schering-Plough Animal Health Corp., Kenilworth, NJ). A venous blood sample was obtained from the rabbit. Both eyes were immediately enucleated, and the aqueous humor of each eye was withdrawn into a syringe, using a 30-gauge needle. The aqueous humor and eyes were then immediately frozen at ⫺80° C. Serum was obtained by allowing the blood sample to clot at room temperature for 1 hour, followed by centrifugation. The serum was frozen at ⫺80° C until tested. Frozen vitreous was then eviscerated whole from the eye and frozen at ⫺80° C until ready to be analyzed. Before analysis, the frozen vitreous was weighed, then defrosted and solubilized in 1.0 ml of 1% bovine serum albumin in 1⫻ phosphate-buffered saline (PBS) on a rotator overnight at 4° C. The following day, the samples were centrifuged at 2000 rpm for 10 minutes. The volume of the sample after centrifugation was measured. All samples were diluted as necessary before the immunoassay.

Vitreous Humor Aqueous Humor

100 10 1 0.1 0.01

0

8

16

24

32

Time (days) Figure 1. Ranibizumab concentration in the vitreous humor and aqueous humor after intravitreal injection of 0.5 mg of ranibizumab. Samples were taken from the aqueous and vitreous of the injected eye. No ranibizumab was detectable in the serum.

Bakri et al 䡠 Pharmacokinetics of Intravitreal Ranibizumab (Lucentis) Table 1. Comparison of the Pharmacokinetics of Bevacizumab and Ranibizumab Intravitreal Drug

Compartment

Bevacizumab, 1.25 mg

Vitreous Aqueous Serum Vitreous Aqueous Serum

Ranibizumab, 0.5 mg

Half-life (Days)

MRT (Days)

Tmax (Days)

4.32 4.88 6.86 2.88 2.84

5.92 6.77 12.8 4.03 6.77

1 3 8 1 3

Cmax (␮g/ml)

% of Vitreous Cmax

400 37.7 9.4 3.33 0.8 162 17.9 11.0 None detected

Exposure to Drug (% of Vitreous Exposure) 8.9 1.6 14.2

Cmax ⫽ maximum concentration; MRT ⫽ mean residence time (time to clear ⬎69% of the drug); Tmax ⫽ time to attain maximum concentration. Samples were taken from the aqueous and vitreous of the injected eye.

Table 1 shows the maximum concentrations of ranibizumab in the aqueous humor and vitreous humor of the injected eye. The maximum concentration of ranibizumab attained in the aqueous humor was 11.0% of the maximum concentration in the vitreous. Taking into account the half-life, the total exposure of the aqueous humor to ranibizumab was 14.2% of that of the vitreous.

Discussion In a previous pharmacokinetic study,12 both eyes of a monkey were injected with 0.5 mg of intravitreal ranibizumab. The half-life of 0.5 mg of ranibizumab was 2.6 days in the vitreous cavity in this monkey model, which is comparable with our half-life of 2.88 days in the rabbit. The maximum serum concentration attained in the monkey was 150 ng/ml, but this was after bilateral intravitreal injection. Therefore, no data were obtained on ocular concentrations in the fellow eye. Maximum concentrations of ranibizumab in the vitreous cavity were 169 ␮g/ml at 6 hours in the monkey study and 162 ␮g/ml at 1 day in our rabbit study. Our study showed no ranibizumab in the serum, but review of the monkey study detected maximum serum concentrations at 3 hours after bilateral intravitreal injection of 0.5 mg of ranibizumab. A study of 4 further rabbits at 3 hours and 3 rabbits at 8 hours showed no ranibizumab in the rabbit serum. It is difficult to compare directly the pharmacokinetics of ranibizumab in this rabbit model with that of ranibizumab in the monkey model. Monkeys have a larger volume of distribution, and in that study, a bilateral injection was performed. However, using the same rabbit model as in our previously published work11 on the pharmacokinetics of intravitreal bevacizumab (Fig 2) allows us to make a direct comparison of the pharmacokinetics of bevacizumab and ranibizumab. It is important to know the difference between intravitreal bevacizumab and ranibizumab with regard to the tissue distribution and clearance, to allow us to make better clinical decisions on the optimal dosing of the drug and the assessment of its systemic safety. This rabbit model is a previously established model used to study intravitreal pharmacokinetics of other drugs.13–15 It allows the simultaneous sampling of aqueous humor, the entire vitreous, and serum at different time points in different rabbits. However, in rabbits the retina is less vascular,

the vitreous cavity is smaller (1.5 ml, vs. 4.5 ml in humans), the lens is larger, and the serum compartment is smaller than in humans. These factors may result in differing pharmacokinetics, as compared with humans. In our bevacizumab pharmacokinetic study, very small amounts of bevacizumab were found in the fellow uninjected eye. Avery et al9 have shown that retinal neovascularization in diabetics responds to very low doses of bevacizumab (6.2 ␮g) injected into that eye. They have reported that some patients with bilateral proliferative diabetic retinopathy show regression of neovascularization in both eyes when injected with intravitreal bevacizumab in only one eye. It would be interesting to note, given our pharmacokinetic comparison data, whether the same fellow eye effects are seen clinically with ranibizumab. Ranibizumab is a smaller molecule (48 kilodalton) than bevacizumab (149 kilodalton). It may therefore penetrate the retina faster and be cleared faster from the systemic circulation than bevacizumab. Avery et al (unpublished work) have shown that ranibizumab penetrates the fullthickness retina at 1 and 3 days in the rabbit, and bevacizumab takes 7 days to penetrate it. In conclusion, after intravitreal injection of 0.5 mg of intravitreal ranibizumab into a rabbit eye, the half-life in the vitreous is 2.88 days, compared with the vitreous half-life of

Figure 2. Bevacizumab concentration in the vitreous humor, aqueous humor, and serum after intravitreal injection of 1.25 mg of bevacizumab. Samples were taken from the aqueous and vitreous of the injected eye.

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Ophthalmology Volume 114, Number 12, December 2007 1.25 mg of intravitreal bevacizumab of 4.32 days. No ranibizumab was detectable in the serum or in the fellow eye, in contrast to intravitreal bevacizumab, which has been detected in the serum and in the fellow uninjected eye.

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