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Keywords:

  • brimonidine;
  • electroretinogram;
  • extended release;
  • intravitreal device

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose:  To evaluate the safety profile of a brimonidine extended release intravitreal implant, in normotensive rabbit eyes.

Methods:  Devices were made from hollow poly-l-lactic acid (PLA) tubes and contained hundred micrograms of brimonidine pamoate. Device was injected intravitreally in one eye of 12 New Zealand pigmented rabbits, whereas other eye was injected with a sham implant in masked fashion. Ocular examination was conducted at baseline and months 1, 3 and 6 including dilated fundus examination and electro-retinogram (ERG). Four rabbits were sacrificed at each time-point for retinal histology. ERG data were compared between groups and time-points using anova.

Results:  No complications were reported from either eye of any rabbits over a 6-month period. Photopic A wave was reduced in the control eye at 1 month compared with baseline (p < 0.01). There was no significant difference in other ERG parameters between the groups at different time-points. Gross retinal histology was normal at all time-points.

Conclusion:  Extended release intravitreal brimonidine device was found to be safe and in normotensive rabbit eyes.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Brimonidine, a α2-adrenergic agonist and an effective intraocular pressure (IOP) lowering agent, has been shown to have neuroprotective effects in various animal studies (Wen et al. 1996; Yoles et al. 1999; Vidal-Sanz et al. 2001; WoldeMussie et al. 2001; Aktas et al. 2010). However, these promising findings in the animal studies have not been replicated so far in the human studies. The lack of consistent intraocular delivery of the drug molecule in high enough dose to the target tissue is among several explanations suggested for this discordance (Danesh-Meyer & Levin 2009; Saylor et al. 2009). Brimonidine delivered extra-ocularly in the form of eye drops may not reach therapeutic levels consistently in the retina where the retinal ganglion cells (RGC) are located. Therefore, development sustained delivery of brimonidine and other neuroprotective agent is vital in the management of glaucoma.

Advances in the polymer technology has led to development of devices made up of bio-degradable polymers such as Poly-L-Lactic Acid (PLA) that can deliver a drug moiety intraocularly over a sustained period at a predetermined release rate (Robinson et al. 2002; Cui et al. 2008; Kim et al. 2008; Bertram et al. 2009). The purpose of this study was to evaluate the safety of a brimonidine impregnated sustained release intravitreal device in rabbit eyes over a period of 6 months by clinical examination, serial ERG and histopathology.

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

All experiments were conducted in accordance with the ARVO statement of the Use of Animals in Ophthalmic and Vision Research and local institutional guidelines. Twelve male New Zealand pigmented rabbits, weighing 2.5–3.0 kg each were included in the study. Animals were housed in separate cages under a 12-hr light/dark cycle with food and water available ad lib. Animals were divided in three groups of four; one group for each time-point of months 1, 3 and 6.

Intravitreal device implantation

Brimonidine impregnated PLA devices and sham PLA devices with no drug in the core were preloaded in an injector with 25-gauge needle for intravitreal injection (pSivida, Watertown, MA, USA). Implants were made from hollow PLA tubes with a diameter of 0.36 mm (small enough to fit into a 25 gauge needle) Brimonidine pamoate was mixed with 10% polyvinyl alcohol (PVA) solution (400 μl PVA solution per 300 mg brimonidine pamoate) and extruded into the PLA tubes. After drying, for 72 hrs, tubes were cut to 3.5 mm in length and the ends were coated with a 10% PVA solution. After further drying, implants were heated at 115°c for 5 hrs and then loaded into the injectors. Each implant appears as a clear tube with white drug core. The injectors were placed in sterilization pouches and sterilized by gamma radiation at 25 kGy ± 10%, by Sterigenics Inc. Each filled PLA implant contained approximately 100 μg of brimonidine pamoate (about 20% of the amount of active found in a single drop of 1% brimonidine, a commercially available eye drop for glaucoma). The PLA tube is biodegradable and is expected to disappear completely from the vitreous cavity over several months. In-vitro tests suggest biodegradability over a period of 12–15 months.

Rabbits were anaesthetized with intramuscular ketamine (25 mg/kg) and Xylazine (2 mg/kg). The devices were injected trans-sclerally under aseptic conditions and the intravitreal location was confirmed with indirect ophthalmoscopy after pupil dilation with 1% tropicamide and 2.5% phenylephrine. A sham device was placed in one eye and brimonidine device was placed in a fellow eye of each rabbit. The experiment was masked and investigators were not informed of the group until the completion of the experiment. The rabbits were examined daily for 5 days after implantation for any evidence of infection from the injection. Rabbits were given topical gentamicin and prednisolone ointment once a day for 5 days.

The rabbits were divided in three groups of four rabbits each: one group for each evaluation time-points at months 1, 3 and 6. Four rabbits were killed at each time-point (1, 3 and 6 months) with intravenous injection of lethal dose of sodium pentothal.

Clinical ophthalmic examination

Ophthalmic examinations were conducted at baseline before device implantation and then at 1 week, and at 1, 3 and 6 months after implantation. The anterior segment examination included evaluation of the cornea, anterior chamber, iris and lens. The vitreous and retina were observed with indirect ophthalmoscope. IOP was recorded at each time-point under topical anaesthesia using Tonopen-Vet (Reichert Inc., Depew, NY, USA).

Electroretinogram

Standard Ganzfeld scotopic (dark-adapted) and photopic (light-adapted) full field electro-retinogram (ERG) signals were obtained at baseline and months 1, 3 and 6 after device implantation from both eyes. Burian-Alan paediatric bipolar corneal contact lens-type electrodes (Hansen Laboratory, Iowa city, IA, USA), containing both reference and active electrode integrated into contact lens-type electrode, were used in each eye with methylcellulose as coupling agent and the ground needle electrode was placed subcutaneously centrally between the ears. Efforts were made to keep conditions uniform for all animals at all time-points. Therefore, the same electrode was used for the right eye and the left eye of each animal every time. ERGs were performed in the morning session, in the same room, at a consistent warm temperature, and rabbit body temperature measured both before and after the ERG. The rabbits were placed on their stomach on a custom built platform and eyes were consistently aligned with the markings located on the headrest. The platform was then slid into the Ganzfeld bowl, aligning the headrest markings to the inner edge of the Ganzfeld bowl to assure consistent amplitude recordings. ERG was recorded from both eyes simultaneously under continuous monitoring for displaced electrode or poor signal.

General anaesthesia was induced as described previously and both pupils were dilated. Signals were amplified at 10 000 gain and band-passed filtered between 0.1 and 1000 Hz. The scotopic ERG was recorded after 1 hr of dark adaptation. The photopic ERG was recorded after light 10 min of continuous light adaptation with background illumination of 34 cd/m2. Scotopic and photopic a-wave and b-wave peak amplitudes were measured from the preresponse baseline and in case of b-wave when a wave was present from a wave maximum. The a-wave or b-wave implicit time was determined from the flash presentation to the start of either the a-wave or the b-wave respectively.

Histology

Eyes were enucleated immediately posteuthanasia and fixed in histochoice solution for 3–4 weeks. Afterwards, eyes were transferred to 70% ethanol and processed for standard histologic tissue preparation. The eyes were embedded in paraffin and sectioned at 2 and 4 mm from the optic nerve in the sagittal/axial orientation. The sections were stained with haematoxylin and eosin (H & E) and evaluated with light microscopy for possible alterations in the retinal morphology secondary to the intravitreal implantation of the device.

Statistical analysis

ERG parameters were compared between the study eye group and the control eye group using one-way analysis of variance (anova) with Bonferroni post-test correction to adjust for multiple comparisons. Statistical analyses were performed using jmp software version 6.0.2 (SAS institute, Cary, NC, USA). A p value < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Clinical observations

After the intravitreal injection of the device, the vitreous appeared clear in both the study eyes and the control eyes. All implants were noticed to have settled inferiorly in the vitreous cavity. No difference in the anterior segment findings was observed between the groups over the 6-month follow-up period. No signs of vitreous inflammation, vitreous haemorrhage, and retinal detachment or optic nerve changes were noticed in any eyes. IOP was maintained within normal limits during the entire study in both groups.

Electroretinogram

Table 1 presents ERG measurements at all study time-points for the study and control eyes. There was no statistically significant difference in any ERG parameters at baseline and months 1, 3 and 6 except B implicit time in the eyes with sham implant. B implicit time was significantly lower at 1 month and 6 months compared with baseline in the control group. As evident from Fig. 1, the ERG waves from both eyes followed very similar pattern. The ERG values were lower in the study eye at all time-points for all animals compared with the control eye; however, this difference was not statistically significant (Fig. 2).

Table 1.   ERG measurements at all study time-points for the study and control eyes.
 Study eyespControl eyesp
Baseline1 month3 months6 monthsBaseline1 month3 months6 months
Photopic
 A wave9.6 (8.2–11.0)8.6 (7.2–10.1)8.1 (6.5–9.8)10.8 (7.7–13.80.5911.0 (9.6–12.5)8.5 (7.1–10.0)9.5 (7.9–11.1)10.6 (8.3–12.9)0.13
 B wave90.2 (77.2–103.2)91.5 (78.5–104.5)85.3 (60.4–100.3)86.4 (70.4–99.4)0.3795.1 (82.0–108.1)92.2 (75.2–105.2)90.6 (72.3–109.3)82.3 (56.3–108.7)0.88
 A implicit time (seconds)11.7 (11.1–12.3)10.9 (10.3–11.5)11.0 (10.2–11.7)10.5 (10.0–11.5)0.8911.4 (10.8–11.0)10.9 (10.3–11.5)10.8 (10.3–11.1)11.7 (10.7–12.7)0.11
 B implicit time (seconds)29.5 (28.7–30.2)29.0 (28.2–30.8)28.7 (27.1–30.3)30.0 (28.3–31.6)0.2130.0 (29.3–30.8)30.0 (29.2–30.7)30.7 (29.9–31.5)30.5 (29.1–31.8)0.16
Scotopic
 A wave72.1 (63.4–80.774.3 (65.6–83)67.0 (56.8–77.3)66.0 (53.8–72.1)0.2481.6 (72.7–90.3)83.9 (75.3–92.6)79.1 (67.2–91.1)74.2 (58.2–98.3)0.18
 B wave213.1 (186.6–239.5)210.4 (189.0–236.9)220.4 (186.0–254.1)221.1 (188.0–244.9)0.06238.5 (212.0–264.9)249.5 (222.5–275.6)274.8 (236.5–313.4)267.6 (208.9–326.2)0.54
 A implicit time (seconds)11.7 (10.9–12.5)11.4 (10.6–12.1)10.7 (9.7–11.7)11.3 (9.9–12.0)0.2311.5 (10.7–12.2)10.9 (10.1–11.6)10.6 (9.9–11.3)11.5 (10.3–12.60.45
 B implicit time (seconds)72.3 (68.1–76.5)65.8 (61.6–70.6)68.8 (63.1–74.4)70.0 (64.4–72.2)0.1273.0 (68.8–77.3)66.3–62.1–70.5)73.2 (69.2–77.2)66.5 (60.8–72.1)0.00
image

Figure 1.  Representative image of the scotopic electroretinogram in the study eye and the control eye.

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image

Figure 2.  Line chart presenting the (A) mean A wave values and (B) mean B wave values from the study group and the control group at different time-points.

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Histology

No obvious retinal abnormality was noticed on histologic evaluation between the study group and control group at any time-points (Fig. 3). No evidence of retinal toxicity or inflammation was evident in any specimens. Some specimens did show retinal detachment that was considered to be postfixation artefacts.

image

Figure 3.  Light microscopy images of retina at 6 months in (A) study eye and (B) control eye using haematoxylin and eosin. The retinal architecture is intact in both eyes. Magnification, × 20.

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This is the first study evaluating the ocular toxicity of the extended release intravitreal brimonidine PLA polymer-based device in the rabbit eyes. The clinical examination, ERG and histology were normal in all study eyes. Furthermore, no significant adverse effect was noted from the procedure of injecting this device intravitreally either in the study or in the control eyes.

This data indicate that extended release intravitreal brimonidine device may be safe for use in the humans. It is important to note that normal ERG does not rule out functional changes at the level of ganglion cell layer or nerve fibre layer. Similarly, global ERG cannot detect focal ERG changes that may occur should the implant rest on the retina and lead to high local levels of brimonidine. Although one cannot extrapolate safety data from rabbits to humans, the long track record of brimonidine use for glaucoma treatment and very low cumulative exposure to the drug from the device in this study makes it very unlikely that the long-term release of brimonidine will be harmful to retina or other ocular structures in humans. In fact, delivery of intravitreal brimonidine using patented posterior segment drug delivery system in humans is ongoing and the results are awaited (NCT00693485 and NCT0108029). We believe PLA intravitreal device has benefits over topical delivery of drugs. This modality allows drug release for up to 3 months at a predetermined rate close to the target tissue. Other similar intravitreal nonbiodegradable devices have been found to be effective and safe for intraocular. (Campochiaro et al. 2010; Haller et al. 2010).

Neuroprotective effects of brimonidine may be mediated via its α2-agonist activity and upregulation of endogenous brain-derived neurotrophic factor (BDNF) in RGCs (Gao et al. 2002). In this study, Gao et al. 2002 found that a single intravitreal injection of low-concentration intravitreal brimonidine was able to significantly increase endogenous BDNF expression temporarily. The elevation of BDNF declined to nonsignificant levels within 1 week after the injection. It may be possible to achieve sustained elevation of endogenous BDNF levels using sustained release PLA device to deliver brimonidine. As BDNF can promote ganglion cell survival, upregulation of endogenous BNDF expression may result in neuroprotection. Further animal studies are necessary to explore this possibility.

Topical application of brimonidine can achieve wide spread intraocular distribution (Acheampong et al. 2002). Iris was the site with the highest concentration of radioactivity following a single topical application of [14C] brimonidine to pigmented rabbit eyes and cynomolgus monkey eyes. Radioactivity was also measured in the posterior segment including vitreous, choroid/retina and optic nerve head. Similarly, the topical application of brimonidine ophthalmic solutions (0.2%) administered in human eyes resulted in vitreous levels well above that required to activate α2-receptors but were hugely variable at 185 nm ± 500 nm (Kent et al. 2001). Furthermore, concentrations were vastly higher in pseudophakic eyes (256 ± 639 nm) and aphakic eyes (164 ± 69 nm) compared with phakic eyes (9.3 ± 8.0 nm), although small number of subjects precluded meaningful statistical comparison. Significant proportion of patients requiring neuroprotective therapy are likely to be phakic, at least during early stages of the therapy and thus may not get benefit of this drug if it is delivered topically. Further, topical application of any medication also has drawback of reliance on patients to instil these drops appropriately and may lead to further variability (Hermann et al. 2011). The placement of an intravitreal sustained release device overcomes these drawbacks and can deliver the drug in a more reliable and consistent fashion.

There are some drawbacks in this study. Firstly, a small number of animals were studied; however, given it was a toxicity study and not efficacy study our belief is that this number is adequate. Secondly, we do not have any data from first 3 weeks after the device placement. The device releases drug at higher level in first 7–10 days, which may have led to some changes in ERG or retina at the earlier time-points. However, even if these changes may have occurred in the early stages of the therapy, they were reversible given that eye examination, ERG and histology were normal at 1-month period. Thirdly, normal ERG and retinal architecture does not rule out possibility of functional changes in RGC, however, proven neuroprotective action of brimonidine makes this possibility unlikely.

In summary, this study found the extended release intravitreal brimonidine impregnated PLA device to be safe in rabbits. This is potentially important mode of delivering brimonidine introcularly that needs to be explored further in human studies.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Phillip Moss for performing electroretinogram and Dr Jaykrishna Ambati for laboratory facilities.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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