Application of in vivo confocal microscopy to the objective evaluation of ocular irritation induced by surfactants.

An ocular irritation test using confocal laser scanning ophthalmoscopy has been developed in which corneal lesions subsequent to instillation of surfactants are specifically marked by fluorescein and assessed by digital image processing. The sum of the observed fluorescent corneal areas is taken into account as an endpoint of ocular irritation. Eight currently used nonionic, cationic and anionic surfactants were applied onto the cornea of rabbits and mice, four times per day during 3 days at various concentrations. Benzalkonium chloride, a cationic surfactant, at a concentration range of 0.01-0.5%, was tested in the same manner. The cornea was evaluated in vivo for ocular tolerance by confocal microscopy. In both rabbits and mice, the test revealed following irritation rankings: cationic>anionic>nonionic surfactants. Furthermore, in both animal models, the ocular damage increased with the concentration of benzalkonium. The test was sensitive enough to detect ocular microlesions at concentrations of surfactants as low as 0.01% for benzalkonium. These findings demonstrate the usefulness of confocal microscopy for the non-invasive, in situ evaluation of ocular tolerance.

Topical use of chitosan in ophthalmology: tolerance assessment and evaluation of precorneal retention.

The mucoadhesive polysaccharide chitosan was evaluated as a potential component in ophthalmic gels for enabling increased precorneal drug residence times. This cationic vehicle was expected to slow down drug elimination by the lacrymal flow both by increasing solution viscosity and by interacting with the negative charges of the mucus. The molecular weight (Mw) and concentration of polysaccharide were studied in four types of chitosan as parameters that might influence ocular tolerability and precorneal residence time of formulations containing tobramycin as therapeutic agent. An ocular irritation test, using confocal laser scanning ophthalmoscopy (CLSO) combined with corneal fluorescein staining, clearly demonstrated the excellent tolerance of chitosan after topical administration onto the corneal surface. Gamma scintigraphic data showed that the clearance of the formulations labelled with 99mTc-DTPA was significantly delayed in the presence of chitosan with respect to the commercial collyrium (Tobrex(R)), regardless of the concentration and of the molecular weight of chitosan in solution. At least a 3-fold increase of the corneal residence time was achieved in the presence of chitosan when compared to Tobrex(R).

Ocular tolerance of preservatives on the murine cornea.

We investigated the effects of instilling 13 commonly used preservatives on the murine cornea in vivo. Due to the instillation of preservatives, micro-lesions are formed on the cornea and can be selectively marked by fluorescein. The sum of the resulting fluorescent areas was measured using an episcopic microscope coupled to an image processing system. All the tested preservatives proved to be well-tolerated by the eye at commonly used concentrations. However, in some cases, increased concentrations of preservatives or combinations resulted in significant increase of the amount of corneal damage. With increasing the concentration, corneal lesion increased the most in the case of cetylpyridinium. While a combination of chlorobutanol 0.5% and phenylethylalcohol 0.5% did not result in an increase in corneal damage (when compared to the use of each separately), the associations of thiomersal 0.02% and phenylethylalcohol 0.4% on one hand and of edetate disodium (EDTA) 0.1% and benzalkonium 0.01% on the other, resulted in significant increases in the amount of corneal damage. However, in none of the tested combinations, the increase in the observed damage exceed the limit of ocular intolerance we had defined beforehand: thus, they were all deemed relatively well-tolerated. In the last part of the study, we investigated the effects of combining several preservatives, at usual concentrations, with an anesthetic solution of oxybuprocaine and found no notable increase in ocular damage.

Dorzolamide, a 40-year wait. From an oral to a topical carbonic anhydrase inhibitor for the treatment of glaucoma.

Dorzolamide, on the basis of its pharmacological profile and lack of undesirable side effects in safety assessment studies together with the fact that it could be formulated in solution at 2%, underwent extensive clinical studies. Early clinical studies in the development of dorzolamide have been described elsewhere (Maren, 1995; Serle and Podos, 1995). In a 1-year study in which a comparison was undertaken in patients for intraocular pressure lowering effects between 2% dorzolamide administered three times daily, 0.5% betaxolol twice daily, and 0.5% timolol twice daily, the peak reductions in intraocular pressure were 23, 21, and 25%, respectively. Tachyphylaxis did not develop to dorzolamide nor were electrolyte and/or systemic side effects encountered (Strahlman et al., 1995). The latter is consistent with results of a pharmacokinetic study in humans in which plasma levels of dorzolamide were lower than the limit of detection (5 ng/ml) at a time when the red blood cell content of dorzolamide had reached steady state which was appreciably less than the red blood cell content of the enzyme (Biollaz et al., 1995). Patients taking 0.5% timolol twice daily received either 2% dorzolamide twice daily or 2% pilocarpine four times daily for 6 months and the additional reductions in intraocular pressure elicited by dorzolamide and pilocarpine were very similar. However, pilocarpine usage resulted in a higher discontinuation rate (Strahlman et al., 1996). In a separate study in which dorzolamide and pilocarpine were compared at these dosage schedules, patients preferred dorzolamide to pilocarpine by a ratio of over 7 to 1 in terms of quality of life (Laibovitz et al., 1995). In summary, the quest for a topical, ocular hypotensive, CA inhibitor, though time-consuming, was a successful one with the introduction of dorzolamide into general clinical practice.

Gamma scintigraphic comparison of eyedrops containing pilocarpine in healthy volunteers.

The aim of the present study was to compare, in healthy human volunteers (male and female), the corneal contact time of various formulations, each containing one viscosity enhancer from the following list: a phase-transition system (gellan gum, Gelrite), a heteropolysaccharide (xanthan gum) and currently used polymers hydroxyethylcellulose, hydroxypropylmethylcellulose, or poly(vinyl alcohol). These different solutions were compared to a reference solution containing no viscosity enhancers. The corneal contact time of the formulations was evaluated over more than 20 minutes by gamma scintigraphy using Technetium-99m (Tc-99m DTPA) as a radioactive label. An eyedrop containing pilocarpine salts (25 microliters) was instilled in one eye only. Each volunteer received 4 formulations, the interval between the instillations being one week. The protocol has been approved by the relevant institutional human experimentation committee. One minute after instillation, only 23% of the reference solution remained on the ocular surface, whereas the novel formulations maintained, respectively, 77% (xanthan gum) or 82% (Gelrite) of the tracer on the ocular surface. Twenty-one min after instillation, 12% (reference solution), 25% (xanthan gum solution), and 39% (gelrite solution) of the tracer remained on the ocular surface. The results confirm that an increase in viscosity of the formulation (xanthan) delays the clearance of the instilled solution by the tear flow. The effect of the gelation mechanism is superior, especially at the later time points. In this respect, xanthan gum and, particularly, Gelrite are suitable vehicles for ophthalmic drugs.

Mucoadhesion of copolymers and mixtures containing polyacrylic acid.

Water-soluble polymers were synthesized from dextran and polyacrylic acid and their ocular mucoadhesion was evaluated. One series had polyacrylic acid grafted onto the polysaccharide backbone of dextran, and another series had dextran grafted onto the polyacrylic acid backbone. Mucoadhesion of these copolymers was investigated using a tensile apparatus and compared with that of polyacrylic acid/dextran mixtures prepared in different proportions. Whatever the copolymer structure, no synergistic effects were seen and mucoadhesion was not markedly increased compared to dextran. The adhesion of copolymers was the same as that of mixtures having a similar polyacrylic acid content and was always less than that of polyacrylic acid alone. Formation of an interpolymer complex occurred at concentrations up to 60% polyacrylic acid, and only above this value did bioadhesion increase above that of dextran. When this complex was dissociated by neutralization of the carboxyl groups of polyacrylic acid, the mucoadhesion of the copolymers and the mixtures was improved. These experiments demonstrated that copolymers and mixtures of dextran and polyacrylic acid did not produce polymers with improved ocular mucoadhesion.

Scintigraphic assessment of an ophthalmic gelling vehicle in man and rabbit.

Studies of the rate of clearance of a gellan gum formulation (Gelrite) radiolabelled by the inclusion of technetium-99m labelled diethylenetriaminepentaacetic acid were carried out in volunteer subjects and in rabbits. Disposition was followed by gamma scintigraphy and compared with 0.5% w/v hydroxyethylcellulose (HEC) solution and isotonic saline administered to the same subjects. Clearance of all solutions was found to follow bi-exponential kinetics with differences in clearance rates between the two species studied. A significant retention of the gellan gum formulation compared to HEC (p = 0.006) or saline (p = 0.009) was noted in man, but not in the rabbit. In this latter species the HEC showed greater retention compared to Gelrite. The species-specific differences in the precorneal residence of the formulations are attributed to the different physiological responses following instillation of solutions into the eye.

Corneal toxicity studies in rabbits and dogs with hydroxyethyl cellulose and benzalkonium chloride.

Hydroxyethyl cellulose (HEC) is used as a viscosity-enhancing agent in ophthalmic formulations to prolong corneal contact time and increase intraocular drug levels. Benzalkonium chloride (BAK) is the preservative most frequently used in ophthalmic formulations. Corneal epithelial changes were seen by slit lamp and light microscopic examination in rabbits but not dogs after multiple instillations of an ophthalmic vehicle containing 0.01% BAK and 0.5% HEC. Microscopically, there was sloughing of superficial epithelial cells and a slight loss of polarity of the basal cells. Formulations with 0.01% BAK and HEC, at concentrations between 0.3 and 0.8%; caused these changes but not with BAK or HEC alone. It was concluded that hydroxyethyl cellulose increased the viscosity and prolonged the contact time of BAK with cornea resulting in corneal epithelial damage in the rabbit. Physiological and anatomical features of the rabbit combined with the increased contact time were concluded to favor these changes in this species. The results confirm that the rabbit is a sensitive and unique species in studies of ocular toxicity of drugs.

Ocular distribution studies of the topical carbonic anhydrase inhibitors L-643,799 and L-650,719 and related alkyl prodrugs.

The ocular distribution of the carbonic anhydrase inhibitors 6-hydroxybenzothiazide-2-sulphonamide (L-643,799) and 6-hydroxybenzothiophene-2-sulphonamide (L-650,719) has been investigated in albino rabbits after conjunctival administration of these compounds or related alkyl prodrugs. The ocular penetration of L-650,719 has been compared in ocular normotensive rabbits and in animals whose intraocular pressure has been experimentally elevated.

Electron-capture GLC assay of dichlorphenamide.

GLC with electron-caputre detection was applied to the assay of the carbonic anhydrase inhibitor dichlorphenamide and demonstrated a sensitivity of 10 ng in 0.5 ml of rabbit serum or whole aqueous humor (congruent to 0.25 ml) from one rabbit eye. After extraction of the drug and internal standard (monochlorphenamide) from the biological fluid, these compounds were converted to their tetramethyl derivatives by a nucleophilic alkylation method. Dichlorphenamide contents of aqueous humor and serum of rabbits treated with this drug are reported.

Preparation, gas chromatography and mass spectrometry of methyl and trimethylsilyl esters of indomethacin.

The preparation and gas chromatographic-mass spectrometric behavior of the methyl and trimethylsilyl esters of indomethacin, 1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid, are described. Reaction of this anti-inflammatory drug with diazomethane or bis(trimethylsilyl)acetamide forms the expected esters. Derivatization with dimethylformamide dimethylacetal yields two compounds, the methyl ester (major product) and a methyl ester-dimethylaminomethylene condensation (at the alpha-carbon of the side chain) product (minor). Experiments with 5-O-desmethyl-indomethacin have demonstrated that using the described diazomethane methylation conditions no alkylation of the phenolic group occurs. Esterification combined with an isolation procedure allows the determination of indomethacin levels in plasma and aqueous humor of rabbits, the 4-fluorobenzoyl analog serving as internal standard. The derivatives exhibit excellent electron capture properties allowing quantitative assay of the drug at the submicrogram level. Precision and accuracy for plasma samples varied from 92 +/- 19% (5 ng/ml) to 96 +/- 1.5% (1000 ng/ml). The analogous values for aqueous humor are superior: 97 +/- 5.6% and 99 +/- 2.2%, resectively.

Comparison of the effects of timolol and other adrenergic agents on intraocular pressure in the rabbit.

The effect of timolol, propranolol, epinephrine, and isoproterenol on intraocular pressure (IOP) (measured by tonometry) were compared after topical administration in conscious rabbits. Epinephrine and isoproterenol decreased IOP in normotensive rabbits, whereas propranolol had no effect. Timolol produced only a slight and inconsistent lowering of IOP in normotensive rabbits. All four agents reduced IOP elevated by an oral water load; the adrenergic agonists were substantially more active than the two beta-adrenergic blocking agents. In alpha-chymotrypsin-induced ocular hypertension, epinephrine, isoproterenol, and timolol were essentially equally effective, whereas propranolol exhibited only weak activity. In this latter model, timolol did not lose its effectiveness after multiple instillations (three/day) over an 8-day period. The concentration of timolol in the acqueous humor after topical application of effective hypotensive doses was relatively high as compared to that found in plasma. In addition, topical doses of timolol required to lower IOP were considerably greater than those needed to reduce or block the ocular hypotensive activity of isoproterenol. The mode of action and therapeutic implications of beta-adrenergic blocking agents in glaucoma are discussed.

Arachidonic acid-induced elevation of intraocular pressure and anti-inflammatory agents.

Arachidonic acid, a precursor of prostaglandin E2 subconjunctivally injected into rabbit eyes in concentrations of 0.8 to 5 per cent, produced a dose-related rise of intraocular pressure (IOP) similar to the one observed after topical administration of PGE2 in concentrations of 0.001 to 0.1 per cent. Indomethacin, a nonsteroidal anti-inflammatory agent, had an inhibitory effect when administered systemically or topically prior to arachidonic acid. Dose-response relationships were demonstrated by topical and oral administration of Indomethacin which, as expected, had no effect on PGE2. Dexamethasone did not exert any inhibitory effect on the increased IOP following arachidonic acid administration. Indomethacin applied topically readily penetrated into the aqueous humor in concentrations considerably above those appearing in the plasma, which justifies the use of this route of administration. Since prostaglandin (PG) appeared to be implicated in ocular inflammation, these findings underline the specificity of action of Indomethacin and suggest its use in the topical treatment of ocular inflammation.