Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

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A.Application of Drug and Analysis of Effect

The delivery of drugs to the posterior pole of the eye by topical application to the cornea faces several challenges, including a long diffusion pathway to the target tissue, corneal impermeability to large molecules, and convectional flow of intraocular fluid in the opposite direction. Protocols designed to examine the effects of drugs delivered to the cornea must therefore demonstrate penetration to the posterior pole and biological efficacy. A recent and careful study by Mizuno et al. [2] to test the effects of nipradilol met these challenges in a study using male Japanese White rabbits.

B.Penetration Studies

Wake rabbits were placed in restraining cages. Radiolabeled [3-14C]-nipradilol (Amersham Pharmacia Biotech) was applied to the lower cul-de-sac of the right eye twice daily (100 L of a 1 % solution) for 7 days. Care was taken to apply the agent at the same times each day. Thirty minutes after the final application, the rabbits were anesthetized with phenobarbital and the eyes enucleated. The basic control for these experiments was to apply saline (or appropriate vehicle) to fellow eyes and to eyes of rabbits that did not receive radiolabeled agent.

Enucleation was performed by the following procedure to minimize contamination of ocular tissues with the radiolabeled nipradilol. An incision was made along the outer edge of the orbit and the eyelids were clamped together with a surgical clip. The entire globe, complete with eyelids and surrounding tissues, was then removed as a pouch. The pouch was then immediately immersed in hexane and solid carbon dioxide for 2 min and then stored at 15°C.

Distribution of the [3-14C]-nipradilol was analyzed by dissecting the tissues of the eye under semi-frozen conditions, solubilizing them, and counting them in a scintillation counter. The actual amount of agent that had penetrated different tissues was calculated from the quench-corrected measurement of radioactivity and the known specific activity of the radiolabeled compound. Note that quench corrections are routinely performed automatically by modern scintillation counters and specific activity should be determined as a function of the half-life and the age of the isotope being used.

Tissues that were analyzed in the Mizuno study included anterior chamber components (cornea, aqueous humor, iris, and lens) and posterior chamber components (vitreous and retina/choroid). After two applications every day for 7 days, the vast majority of labeled nipradilol was localized to the cornea (1284fold over controls) and iris, with moderate levels being detected in the aqueous humor. Substantially less nipradilol was detected in the lens, vitreous, and retina/ choroid (threefold over controls), but the levels in the retina were still significantly higher than in control eyes. Also, the effective concentration of nipradilol

in this tissue was estimated at 0.12 M, which was in the clinically effective range of the drug.

C. Efficacy Studies

In many studies, efficacy is measured as the ability to prevent loss of target cells in the retina. It is important to note, however, that topically applied drugs could achieve these effects through systemic means (i.e., affecting ocular blood flow) rather than by directly acting on the target cells. To test the physiological effect of this dose of nipradilol, Mizuno et al. utilized a bioassay that took advantage of the antagonist effect of nipradilol on endothelin-1. Nipradilol has a vasodilating activity, whereas endothelin-1 has a vasoconstricting effect. Fundus photography was utilized to determine the diameter of the two major retinal arteries at the rim of the optic nerve head. The change in artery diameter was monitored with successive fundus photographs after an intravitreal injection of 20 L of 5 10 8 M endothelin-1. These experiments showed that topical application of nipradilol provided a significant reduction in the vasoconstriction effects on intravitreally injected endothelin-1 up to 60 min, indicating that sufficient amounts of the topically applied drug had penetrated to target endothelial cells at the back of the eye.

D.The Route of Trans-Corneal Delivery to the Back of the Eye

Radiolabeled-drug studies suggest that the route of drugs administered to the cornea to the back of the eye is periocular, rather than through diffusion across the cornea and on into the vitreous. The evidence indicating this is the very low level of accumulation of drug in the vitreous. In addition, nipradilol (molecular weight of 326.35) is small enough to penetrate through the sclera, where the permeability constant is inversely proportional to the molecular weight for solutes between 285 and 70,000 daltons (see Sec. IV). Additionally, studies have shown that both timolol and betaxolol accumulate in the connective tissue of Tenon’s capsule in patients with prolonged usage of these drugs as topical drops [3], supporting this mode of access to the posterior pole.

III. INTRAVITREAL DELIVERY

Intravitreal application is the most direct method for delivering a compound to the cells of the retina. It has been used to test recombinant viruses carrying neuroprotective genes [4], recombinant proteins such as growth factors [5–7], and small compounds such as flupirtine [8].

A.Intravitreal Injections

Injections into the vitreous are relatively straightforward and in most animals can be accomplished with a suitably small gauge needle. Injections are always made in anesthetized animals. Rats can be injected with a 27–30 G needle with little difficulty. Mouse eyes are more difficult to inject into and require a two-stage process of first piercing the conjunctiva and sclera with a 30-G needle followed by passing a glass micropipette through the hole into the eye [9]. Ideally the micropipette should be pulled mechanically using Drummond borosillicate glass capillary tubing (Cat. No. 3-000-210-G/ID 0.704 mm). Injections are easiest using needles with a long tapered point rather than a steep point typical of pipettes used for single cell injections or electrophysiology. Hand-pulled glass pipettes can also be used successfully. A total maximum volume of 2 L can be injected into the mouse eye without damaging the retina. There are reports that up to 10L injections can be made into the rat eye.

There are several disadvantages to intravitreal injections. The first is the transient time of exposure to the drug, since the turnover rate of fluids (and hence clearance of a drug) in the vitreous is quite high [1]. This may necessitate repeated injections, which are not trivial on small eyes, although, such have been performed successfully in the rat eye for flupirtine [8]. Repeated injections also run the risk of a variety of unwanted side effects, including cataract formation, endophthalmitis, retinal detachment, and vitreal hemorrhage [l0]. Alternatively, sustained-release devices have been implanted into the vitreous to allow a more continuous exposure of small molecules [11], and osmotic mini-pumps have been used successfully to provide continuous application of larger molecules such as brain-derived neurotrophic factor [12]. If the neuroprotective agent is a protein, long-term delivery can also be achieved by introduction of a transgene for the molecule into surrogate retinal cells by recombinant viral gene therapy (see Chapter 10). A second problem with intravitreal injections is leakage of the injected fluid from the injection site. This is particularly true in the mouse eye if the initial puncture wound is too large, resulting in a poor seal around the glass pipette. Last, great care must be taken not to damage anterior chamber structures, including the lens, with the injection needle. This can significantly complicate results by secondarily creating a neuroprotective environment in the eye [13]. Lens damage is often marked by cataract formation a few days after the injection and affected animals should be excluded from the data set in experiments employing intravitreal injections. In the mouse, making the initial puncture at a site just posterior to the limbus and then inserting the micropipette used for the injection at a relatively steep angle can avoid this damage. Care must then be taken to avoid penetrating the retina. It is recommended that initial attempts be made using a tracer dye to determine the success of the injections. With practice, it is possible to achieve highly consistent results.

IV. TRANS-SCLERAL DELIVERY

Trans-scleral delivery of compounds is gaining favor as a method of applying neuroprotective agents to the retina, because compounds can be delivered to specific regions of the posterior pole without the complications of repeated intravitreal injections or the requirement for applying high concentrations of drug necessary for both trans-corneal and systemic delivery methods. The sclera is an elastic tissue composed of collagen fibrils and proteoglycans, which has a highly porous nature. Several in vitro diffusion studies using isolated pieces of sclera indicate that the rate of diffusion across this tissue is inversely proportional to the molecular weight of compounds between 285 and 70,000 molecular weight [1,14]). The molecular radius of molecules also influences diffusion, such that more globular compounds diffuse at a higher rate than linear structures of the same molecular mass [10]. More recent studies indicate that even large biomolecules, such as immunoglobulins, can penetrate the sclera [15] and retain their biological activity [16].

Methods of applying compounds to the eye for scleral diffusion fall into two categories depending on the amount and time course of drug delivery required. For short, one-time applications, drug is injected subconjunctivally as a single bolus. Diffusion can occur over a time period of minutes, but this rate can be influenced by factors such as molecular mass, as indicated above, and by scleral thickness, which varies from being relatively thin at the equator of a human eye (averaging 0.39 mm) to relatively thick (1.0 mm) at the optic nerve [17]. Prolonged drug release can be achieved by using a sustained-release method. Like intravitreal delivery, sustained-release methods range from osmotic pumps to preparations of drug in slow-release matrices such as cyanoacrylate adhesive. In addition, Tenon’s fibroblasts are readily transduced with recombinant DNA using adenoviruses [18] or naked plasmids absorbed into a collagen shield [19], methods that can be used to deliver genes encoding proteins small enough to penetrate the sclera. Because the transduced fibroblasts have been shown to express a transgene for several weeks post infection, this method may be used to achieve a biological alternative to a sustained-release device. Adenovirus infection can be achieved simply by a sub-conjunctival injection of virus, although this method often results in a diffuse spread of the infection around the globe (Fig. 1). A more discrete area of transduction can be achieved by soaking a cellulose sponge with virus and placing it under a flap of the Tenon’s capsule and conjunctiva in the region of interest. Significant levels of transduction are obtained using as little as a 5-min exposure. Like any other sustained-release device, the drawback with the gene transfer method is that it is not permanent and the application of recombinant virus would have to be repeated after between 14 and 30 days, when transgene expression becomes quiescent and/or the transduced cells are eliminated. Times of persistence may vary depending on the nature of the transgene

Figure 1 Gross morphology of an enucleated rabbit eye (OD) showing distribution of a recombinant adenovirus injected subconjunctivally 4 days previously. The superior part of the eye is indicated with a suture in the cornea and temporal is to the left of the micrograph. A bolus of 100 µL (approximately 1010 virus particles) of recombinant adenovirus carrying the reporter gene β-galactosidase was injected by introducing a needle under the temporal conjunctiva and working it superiorly to the point indicated by the arrow. After 4 days, the animal was sacrificed and the eye enucleated, fixed briefly in paraformaldehyde and then stained for β-galac- tosidase activity. Stained cells are evident along the area of injection and needle track, while cells in the inferior nasal conjunctiva are not transduced. Histological analysis of transduced conjunctiva show that Tenon’s fibroblasts are readily transduced by adenovirus and therefore may be suitable targets for gene therapy using secreted small proteins that could diffuse across the sclera.

and the promoter being used to drive expression. In addition, this method must employ a transgene construct that allows for secretion of the target molecule.

V.SYSTEMIC DELIVERY

Several systemic routes of drug delivery have been used successfully in ocular neuroprotection studies, including intraperitoneal or intravenous injections and oral delivery. Other modes of delivery include subcutaneous and intramuscular

injections. In general, systemic delivery is the preferred method for protocols that require repeated applications, although this requires the use of high concentrations of drug, which may have undesired side effects. Detailed descriptions of systemic delivery methods for different animals can be found in the appropriate volume of the Laboratory Animal Pocket Reference series. Methods used on rats and mice are described here.

A. Oral Delivery

One of the most dramatic uses of oral delivery for a neuroprotective agent was reported recently by Neufeld and colleagues [20]. In this study, the nitric oxide synthase inhibitor aminoguanidine was introduced into the drinking water of rats, half of whom had been made ocular hypertensive using the vortex vein occlusion method. The amount of drug taken in by each animal was determined by measuring the amount of water consumed. In this particular paradigm, the water was changed 3 times a week, and the rats were kept on the drug for a period of 6 months. Based on the concentration of drug put into the water (2.0 g/L) and the amount consumed (in mL/day), the authors estimated that the rats consumed about 60 mg of drug per day.

Although this approach was successfully used in this study, drawbacks are several-fold. First, it is limited to drugs that are water-soluble. Second, it is important that the drug does not impair the behavior of the animal’s drinking habits, primarily by making the water unpalatable. This was a concern in the Neufeld study, which found that rats consuming aminoguanidine drank an average of approximately 25% less water than rats drinking untreated water, although this difference was not statistically significant based on the variation in the data set. Third, this method may be prone to overestimating the amount of drug consumed by not accounting for gravity-induced seepage of water from the drinking bottle. In a simple experiment testing for gravity flow from standard feeding bottles, it was estimated that 1–2 mL of water is lost per day in conditions where the bottles were undisturbed. It is likely that this loss is greater when animals are agitating the nozzles during drinking.

More controlled drug studies interested in testing the use of oral delivery employ the gavage technique [21]. In this method, a drug is taken up as a metered dose in a syringe, which is then attached to a standard feeding or gavage needle with a blunt tip. In mice, it is possible to use a 30–22 G needle, either straight or curved depending on the size of the mouse, attached to a tuberculin syringe. Up to 100 L can be introduced into the stomach of even small mice.

To feed a mouse, it is essential that the animal is firmly restrained with complete immobilization of the head. The unanesthetized animal is fed by grasping it dorsally and articulating its head down and away from the holder to open the airway and esophagus. The needle is inserted down the esophagus into the