The ocular pharmacokinetics of ketanserin and its metabolite, ketanserinol, in albino rabbits.

Ketanserin, a hypotensive drug with 5-HT2 receptor antagonism, when administered by topical infusion of a 0.25% w/v solution by corneal and scleral applications, was found to lower intraocular pressure with four times more activity than its metabolite, ketanserinol. Drug and metabolite were measured periodically in the corneal epithelium, corneal stroma/endothelium, aqueous humor, iris/ciliary body, conjunctiva, sclera and lens during the infusion period (0-120 min) and the postinfusion period (120-240 min) using a fluorometric reversed-phase HPLC assay developed and verified for the research. The infusion results showed that drug entered the eye by both the corneal and scleral routes. Lateral diffusion occurred between the conjunctiva and corneal epithelium. Drug and metabolite were also detected in the untreated fellow eyes, suggesting contralateral systemic redistribution. In vitro metabolism was studied and found to occur in the corneal epithelium, iris/ciliary body and bulbar and palpebral conjunctiva but not in the corneal stroma/endothelium, aqueous humor and sclera. From noncompartmental analysis, zero-order infusion rate constants, first-order absorption constants, mean residence time, volumes of distribution at steady state (Vss) and clearance (Cle) were obtained using equations specific to the topical infusion method. Vss and Cle of aqueous humor (0.972 ml and 13.55 microl/min) were greater than aqueous humor volume (0.311 ml) and turnover rate (4.7 microl/min).

Uptake of ibuprofen, indomethacin and ketoprofen into isolated rabbit parietal cells.

In the United States and other countries, non-steroidal anti-inflammatory drugs (NSAIDs) can be purchased without a prescription. Due to their widespread use, the drugs' most common side effect, gastric toxicity, becomes a more serious concern. Gastric toxicity can occur directly by contact with mucous membranes or indirectly by the inhibition of prostaglandin production in the gastric mucosa. We have studied the uptake of NSAIDs in gastric tissue, specifically parietal cells removed from the rabbit stomach. New Zealand White rabbits were killed and then used to harvest parietal cells. The purified cells were used to study the uptake of ibuprofen, indomethacin and ketoprofen (NSAIDs) over time and under different experimental conditions. The effects of concentration were investigated for all three NSAIDs. In addition, indomethacin and ibuprofen were used to investigate the mechanism of uptake. Studies were determined for the effects of varied extracellular pH from pH 6 to 8, and inhibitory conditions from depressed temperature (5 degrees C), metabolic inhibitors (sodium azide and 2,4-dinitro-phenol), an ionophore (nigericin) and a sodium free support medium. The interaction of NSAIDs with lysed parietal cells was investigated also. Initial rate data indicated that Michaelis-Menten kinetics were apparent; however, poor solubility of all three NSAIDs prevented complete characterization of the inclusion of a passive transport mechanism. Uptake showed a statistically significant increase (P = 0.01 to 0.05) as pH decreased, also suggesting contribution from a passive mechanism. Studies with inhibitors showed minimal effects. However, uptake at equilibrium for the ionophore, nigericin, showed a 10-fold increase over the control for ibuprofen (P = 0.005) and a 1.4-fold increase for indomethacin (P=0.04). Depressed temperature (5 degrees C) increased the initial rate and uptake at equilibrium 2.1- and 2.2-fold, respectively, for ibuprofen (P < 0.01). For indomethacin depressed temperature increased the initial rate and uptake at equilibrium 2.7- and 5.2-fold, respectively (P < 0.01). The increases at 5 degrees C suggests that adsorption may be an important uptake component. Experiments with lysed parietal cells showed a non-specific uptake phenomenon, suggesting an adsorption component was occurring also.

In vitro and ex vivo hydrolysis rates of ethacrynate esters and their relationship to intraocular pressure in the rabbit eye.

Esters of ethacrynic acid and partial structural analogs were synthesized and evaluated for topical antiglaucoma activity in rabbits. Maximum activity was shown by analogs 2 and 6 (34% and 30% reduction in intraocular pressure recovery rate, respectively). Among the esters, only the ethyl ester (2) was found to be active; the methyl and n-propyl esters (1 and 3) were inactive. Analogs 1-3 were subjected to an estimation of physicochemical properties and chemical stability. However, no correlation was found to exist between the biological activity/inactivity and the physicochemical properties of the analogs. The analogs were evaluated for ex vivo hydrolysis using rabbit aqueous humor (AH), corneal (C) homogenate and iris-ciliary body (ICB) homogenate. For all tissues, the rate of enzymatic hydrolysis increased significantly with an increasing ester chain length. The ICB-mediated hydrolysis was the fastest among the three tissues for all of the analogs. The relationship between the rate constants for the tissue-mediated hydrolyses were: analog 1, ICB>C>AH; analog 2, ICB>C=AH and analog 3, ICB>AH>C. Apparent Michaelis-Menten kinetic parameters were determined for the three analogs using corneal homogenate. Analog 2 showed the highest v0 for all substrate concentrations studied. The conventional Michaelis-Menten equation did not fit the data as well as a sigmoidal model. Both fits of the data showed the fastest enzyme-mediated hydrolysis for analog 2. The parameters of the sigmoidal fit of the data correlated with the activity/inactivity of the analogs. The data indicate that the major factors responsible for the observed activity/inactivity are the differences in the corneal enzymatic hydrolysis of the esters in conjunction with the rapid dynamics of ocular prodrug absorption.

Significance of melanin binding and metabolism in the activity of 5-acetoxyacetylimino-4-methyl-delta2-1,3,4,-thiadiazolin e-2-sulfonamide.

5-Acetoxyacetylimino-4-methyl-delta2-1,3,4,-thiadiazoline -2-sulfonamide (compound (1)) is an ester prodrug that lowered intraocular pressure (IOP) in albino New Zealand rabbits, but was found to be inactive in pigmented Dutch Belt rabbits. In order to explain the differences in pharmacological activity for the two rabbit species, metabolism and melanin binding were studied. Depending on the initial concentration, the binding of compound (1) to natural melanin (Sepia officinalis) was 20-60%. The binding constant, K, at 37 degrees C was 4.32 x 10(5) M(-1) and the maximum moles bound to melanin, r(max), was 4.5 x 10(-7) mol/mg of melanin. From a determination of binding at temperatures between 25 degrees C and 47 degrees C, a van't Hoff plot was constructed to determine enthalpy and entropy changes accompanying the binding process, deltaH and deltaS, respectively. Values calculated from the plot were -12.7 and -15.4 kcal/(mol deg), respectively. Negative values for these parameters are consistent with charge transfer interactions and therefore suggest that this may be an operative mechanism between compound (1) and melanin. The in vitro incubation of compound (1) was also studied with various ocular tissues from both albino and pigmented rabbits which were iris-ciliary body, intact cornea, stroma/endothelium and aqueous humor. A major metabolite, MET 1, was identified and also observed from in vivo analyses of the same tissues following topical application. The metabolite was isolated and subjected to mass spectroscopy and proton nuclear magnetic resonance spectroscopy analysis. From these analyses, it was hypothesized that the formation of MET 1 involved a GSH conjugation mechanism which displaced the sufonamide (-SO2NH2) group. The metabolism was found to be less extensive in the pigmented rabbit than in the albino rabbit and suggested that the binding affinity of compound (1) for melanin was a better explanation for the lack of IOP activity in the pigmented rabbit than differences in metabolism.

Uptake of N,N-dimethyl-2-phenylethylene HCl into acini cells removed from rabbit lacrimal glands.

Acini cells were obtained from the lacrimal gland of the white New Zealand rabbit. Following isolation and purification, the cells were used to study the uptake of N,N'-dimethyl-2-phenylethylamine HCl (AF2975), which was found to be sodium- and proton-independent, but energy-dependent. Uptake was mainly accomplished via a carrier-mediated transport system for which a Km of 8.72+/-0.96 mM, a Vmax of 602.6+/-41.3 nmol/mg of protein/min, and an exponential coefficient of 2.55+/-0.46 were obtained following a least squares nonlinear fit to the Hill equation. With the addition of the metabolic inhibitors, sodium azide or 2,4-dinitrophenol, the initial uptake rates were reduced from the control experiments by 35.7% and 26.2%, respectively.

The influence of tear proteins on the film stability of rabbit tear extracts.

This study was undertaken to gain an understanding of the significance of tear proteins in stabilizing the tear film. Either a sigma agonist, N,N-dimethyl-2-phenylethylamine HCl (AF2975), or a sigma antagonist, haloperidol, was administered to rabbit eyes in order to increase or decrease protein secretion, respectively. At 0, 10 and 60 minutes after instillation, tear proteins were extracted from Schirmer strips and measured for total protein. A portion of the extract was used for separating five major protein fractions using size-exclusion HPLC. Total protein extract or individual protein fractions were measured for surface tension by the horizontal capillary method and for in vitro break up time (in vitro BUT), a newly designed procedure. A statistically significant decrease was measured for surface tension and a concomitant increase was measured for in vitro BUT for the total protein samples at 10 and 60 minutes after instillation of AF2975 compared to the vehicle treated eye. The results for haloperidol yielded an increase in surface tension and an decrease in in vitro BUT. When the tear proteins were separated into five major fractions, only the 23 minute protein fraction was found to decrease surface tension and increase in vitro BUT following AF2975 administration. Haloperidol, a sigma antagonist, showed an exact opposite effect for the total protein and the 23 minute protein fraction.

Tear film stability of protein extracts from dry eye patients administered a sigma agonist.

Fourteen dry eye volunteers placed one to two drops of 0.15% AF2975 (N,N-dimethyl-2-phenylethylamine HCl) in one eye and the vehicle in their other eye four times a day for 21 days. AF2975 is a sigma agonist known to stimulate the release of tear proteins after instillation in rabbit eyes and was tested for its ability to stabilize protein film extracted from dry eye volunteers. After day 7 and again after day 21, Schirmer test strips were inserted in each eye for 5 minutes, measured for wetting, and stored at -20 degrees C for protein analysis. A volume of 600 microliters was used to extract total protein. A portion of the extract was analyzed for total protein. The remainder was used to measure surface tension, to determine in vitro break up time (in vitro BUT) in a newly designed apparatus, and to further analyze for tear lipocalin, formerly known as presystemic tear albumin. Statistically significant differences were obtained between the drug treated eye and the vehicle treated eye for measurements determined for days 7 and 21. Tear extracts from the drug treated eye showed statistically significant decreases in surface tension and increases in in vitro BUT. Extracts from the drug treated eye also showed statistically significant increases in protein content and tear lipocalin. The results suggest that AF2975 may be able to stabilize the tear film by increasing the concentration of proteins in human tears.

Penetration into the anterior chamber via the conjunctival/scleral pathway.

The importance of the conjunctival/scleral pathway as a route of entry into the ciliary body, and in particular uptake and deposition by vessels, was investigated. A constant concentration of methazolamide analogs as well as 6-carboxyfluorescein (6-CB) and rhodamine B (RB) was maintained on either the cornea or the conjunctiva/sclera tissue, the latter excluding the cornea. The solutions were applied with the use of a cylindrical well affixed to the cornea of an anesthetized white rabbit. After two hours, concentrations of drug or dye were measured in cornea, aqueous humor or iris/ciliary body for both routes of entry. Confocal microscopy methods were used to determine reflected fluorescence images for 6-CB and RB. Carbonic anhydrase inhibition, partitioning, solubility and intraocular pressure (IOP) measurements were also determined. Permeability calculations were estimated for drug diffusing against aqueous flow within the posterior chamber. The conjunctival/scleral route of entry produced higher iris/ciliary body concentrations for all compounds except for the lipophilic RB. Confocal microscopy results suggested that drug is gaining entry into the ciliary body through vessel uptake in the sclera. Following entry of drug into the conjunctival/scleral tissue, a significant portion enters scleral vessels and deposits within the ciliary body. Calculations are given that indicate that once drug penetrates the cornea it is highly unlikely drug diffuses through the pupil against aqueous flow to enter the posterior chamber and reach the ciliary body.

The effects of sigma ligands on protein release from lacrimal acinar cells: a potential agonist/antagonist assay.

Sigma receptor antagonists have been proposed as leading clinical candidates for use in various psychotic disorders. Prior to clinical testing, it is imperative that a new agent be correctly identified as an antagonist and not an agonist since the latter may worsen the psychosis. For sigma-ligands many behavioral and pharmacological assays have been developed in an attempt to classify agonist/antagonist activity. These assays evaluate a response or a behavior in an animal model that can be related to clinical efficacy. However, is the action by the presumed antagonist a consequence of sigma-receptor activity? Previously we have identified sigma-receptors in acinar cells of the main lacrimal gland of the New Zealand white rabbit and have measured protein release after the addition of various N,N-disubstituted phenylalkylamine derivatives known to be sigma-ligands by receptor binding studies. Although protein release from acinar cells has been attributed to either muscarinic or alpha-adrenergic stimulation, protein release from sigma-receptor stimulation was also confirmed. In the reported studies here, we isolated and incubated acinar cells with varying concentrations of known sigma-ligands and measured protein concentration. A knowledge of the receptor profile for the disubstituted phenylalkylamines permitted experiments to be designed in which various alpha, muscarinic, serotonergic, and dopaminergic antagonists could be added in equimolar concentrations. Under the conditions of these experiments, statistically significant increases in protein release for sigma-ligands could be attributed to stimulation of sigma-receptors. Haloperidol, an apparent sigma-antagonist, caused a statistically significant decrease in protein release and also inhibited protein release when tested with a known sigma-ligand, AF2975 [N,N-dimethyl-2-phenylethylamine]. In this system, stimulation and inhibition of protein release were defined as agonist and antagonist behavior, respectively. Of particular interest were the results for BMY14802 and +/- pentazocine, both of which were found to be agonists. Various antipsychotic and antidepressant drugs were measured for their agonist/antagonist behavior. Because of multireceptors present in acini, their agonist or antagonist behaviour could not be attributed solely to interaction with the sigma-receptor unless specific antagonists were added.

Determination of tolerance to tear protein release following a twice a day topical application of N,N-dimethyl-2-phenylethylamine HCl.

In an effort to develop a long-term topically active tear stimulant, it was important to determine if tolerance developed following the repeated instillation of N,N-Dimethyl-2-phenylethylamine hydrochloride (AF2975) to the albino rabbit eye. New Zealand white rabbits were administered AF2975 (0.15%) twice a day (9 am and 4:30 pm) for 10 days. The right eye received the drug solution (50 microliters) and the left eye received an equal volume of the vehicle. Prior to dosing and at the end of first and last dose (10 and 60 minutes post-dosing), protein secretion was measured with the use of Schirmer tear test strips placed under the lower lid of each eye for five minutes. The strips absorbed tears from which protein was extracted. Eyes treated with AF2975 showed a statistically significant % increase in protein release compared to baseline values. Control eyes did not show statistically significant increases over baseline. A comparison of % changes from baseline in protein secretion rates after the first and last dose showed no significant differences in either treated or control eyes at 10 and 60 minutes postdosing. These results indicate that tolerance does not occur for protein secretion of topically administered AF2975 (0.15%) following a twice a day dosing schedule for 10 days to the rabbit eye.

Formulation, in vitro dissolution, and ocular bioavailability of high- and low-melting phenylephrine oxazolidines.

The in vitro dissolution and the relative ocular bioavailability of high- and low-melting phenylephrine oxazolidines (HMP and LMP) from a nonaqueous suspension (silicone fluid) were compared. Stability-indicating HPLC assays were developed for evaluation of the prototype formulations, in which a normal-phase HPLC method was necessary for analysis of PO, while a reverse-phase HPLC method was required for analysis of the primary degradation product, phenylephrine (PE), following its separation from the formulation using a short silica gel column. PO was formulated as an ophthalmic suspension in silicone fluid (20 cs) because of its property of undergoing rapid hydrolysis in aqueous media. An experimental test system for measuring the dissolution characteristics of a water-immiscible multiparticulate suspension was designed to obtain the dissolution profiles of suspensions of HMP and LMP. The dissolution rates, which were nearly identical for LMP and HMP, were obtained assuming a quasi-infinite reservoir. A reverse-phase HPLC assay with fluorescence detection was used for measuring the concentrations of PE in aqueous humor and corneal samples. Statistical analysis of the bioavailability data showed that suspensions containing HMP and LMP were equal in extent of absorption following a single topical application to the rabbit eye. The results correlated well with the in vitro dissolution rates of the suspensions of HMP and LMP.

Physicochemical characterization of high- and low-melting phenylephrine oxazolidines.

Phenylephrine oxazolidine is a new prodrug of phenylephrine developed for improving ocular absorption and reducing systemic side effects. In the present study, high- and low-melting phenylephrine oxazolidines (HMP and LMP) were characterized in terms of their stereochemistry and crystal properties. It was found that the molecular configuration of the prodrug in the crystals of either HMP or LMP is identical (5R/2R). The two crystals were shown to have the same IR spectra and X-ray diffraction patterns but different crystal habits, thermal properties, solubilities and intrinsic dissolution rates. Single crystal X-ray structure analysis indicates that crystals of both HMP and LMP are orthorhombic and belong to the P2(1)2(1)2(1) space group with four molecules in a unit cell (a = 20.697 A, b = 7.065 A, and c = 9.304 A). The molecules in the crystal are held together by an intermolecular hydrogen bonding interaction between N(3) and O(13). The different physical properties observed for LMP result from crystal imperfections caused by the presence of trace amounts (often at levels < 0.5%) of an unidentified, structurally related synthetic impurity which can be dispersed in the prodrug. It was observed that both HMP and LMP can sustain thermal and mechanical treatment in the solid state. However, LMP was partially converted to HMP when suspended in certain solvents.

The effect of reduced eyedrop size and eyelid closure on the therapeutic index of phenylephrine.

In this study we examined the relative effects of reducing eyedrop size (from 30 microliters to 10 microliters) and eyelid closure on the ocular efficacy and systemic absorption of 10% phenylephrine. Thirteen subjects participated in a quadruple crossover study that involved dilation with a 10-microliters and a 30-microliters drop of phenylephrine with and without eyelid closure. The 10-microliters drop was just as effective for pupillary dilation as the 30-microliters drop. Eyelid closure improved dilation for both drop sizes. Both eyelid closure and reducing the drug volume decreased systemic absorption of phenylephrine as measured by plasma concentration. When used together, eyelid closure and the smaller drop size reduced plasma concentration by 45%. The therapeutic index for 10% phenylephrine appears to be improved by using a 10-microliters drop followed by eyelid closure.

Lacrimal secretion stimulants: sigma receptors and drug implications.

3H-DTG (1.3-di(2-[5-3H]tolyl)guanidine) or 3H-haloperidol was added to sigma-receptors (25 nM) in the presence of 25 nM spiperone and incubated with increasing concentrations of bromhexine derivatives (phenylalkylamines; 10(-9) to 10(-2)M) in membrane homogenate suspensions. IC50 values for two derivatives ranged from 3.2 to 8.8 nM for both radioligands. A preferred derivative, 7A (N,N'-dimethyl-2-phenyl-ethylamine), yielded an IC50 of 7.8 nM for 3H-haloperidol but showed much less affinity in displacing 3H-DTG (IC50 = 900 nM). Applying the technic of Bromberg [Exp. Eye Res., 40:313-320, 1985], in vitro protein secretion rates were measured following stimulation of either lacrimal gland slices or isolated, intact lacrimocytes with the compounds. In vitro protein secretion rates exhibit a dose-response relationship with increases in protein release up to a concentration of 10(-8) to 10(-4) M for various derivatives of bromhexine and 10(-4) M for carbachol. By means of Schirmer strips, tear fluid was collected over a five minute period at 10 and 60 minutes post-dosing following the topical application (50 microliters) to the right eye of New Zealand white rabbits (n = 20-24) of 7A at various concentrations. Incubation of lacrimocytes with 7A alone (10(-4) M), with haloperidol (10(-4) M) alone or in combination show that 7A is acting as an agonist to stimulate protein release, whereas haloperidol is acting as an antagonist to inhibit release. In vivo protein secretion rates also show a dose-response curve (at both 10 and 60 minutes post-dosing) for 7A that reach a statistically significant maximum in the dosed eye at a concentration of 0.15% w/v. Analysis of protein extracts using size exclusion HPLC shows an increase in secretory proteins, particularly tear-specific prealbumin.

The presence of sigma-receptors in the lacrimal gland.

Applying the technic of S.A. Wolfe et al. [Endocrinology, 124, 1160-1172, 1989], we have established the presence of sigma receptors in isolated, but intact, lacrimocytes excised from main lacrimal gland tissue of the New Zealand white rabbit. 3H-Haloperidol was used as the ligand (0.5-2500 nM), in the presence of spiperone. From a Scatchard plot, a single binding site was statistically chosen over two sites for a majority of the data associated with the intact lacrimocytes. Kd (71.0 +/- 46.4 nM) and Bmax (588.2 +/- 166.7 fmol/mg of protein) values showed lower binding affinity but a similar density of sigma sites in rabbit lacrimocytes when compared to published results obtained for rat exocrine glands and brain tissue. Using the technic of McCann and Su [J. Pharm. Exp. Therap., 257, 547, 1991], membrane suspensions of the sigma receptor were also prepared and tested for binding to radioligands, 3H-DTG, as well as 3H-haloperidol. A Scatchard plot revealed two binding sites for 3H-DTG and one binding site for 3H-haloperidol. The high affinity site for 3H-DTG yielded a Kd of 1.04 +/- 0.64 nM, whereas, Bmax was 135.9 +/- 11.62 fmol/mg of protein. The low affinity site gave a Kd = 75.3 +/- 26.8 nM and Bmax = 344.0 +/- 222.0 fmol/mg of protein. The weaker site is suspected to be intracellular. IC50 values were determined for N,N-disubstituted arylphenylalkylamines (Kd approximately low nM).

Biopharmaceutical evaluation of ibufenac, ibuprofen, and their hydroxyethoxy analogs in the rabbit eye.

Two new structural analogs, 2-(4-hydroxyethoxyphenyl)acetic acid [R3] and 2-(4-hydroxyethoxyphenyl)propionic acid [R4], along with their parent compounds, ibufenac and ibuprofen, were evaluated for their biopharmaceutical properties. The analogs represented substitution of the lipophilic isobutyl side chains of ibufenac and ibuprofen with hydrophilic hydroxyethoxy side chains. Anti-inflammatory activity was evaluated by administering drugs topically to inhibit inflammation induced by using either clove oil or arachidonic acid. The rank order of activity was ibufenac approximately equal to ibuprofen > R3 approximately equal to R4. The new compounds, R3 and R4, were highly water soluble (> 60-fold) and partitioned less (< 1/1500-fold) into the lipid phase when compared to ibufenac and ibuprofen. R3 and R4 each had apparent corneal permeability coefficients of 6 x 10(-6) cm/sec, whereas ibufenac and ibuprofen yielded values of about 22 x 10(-6) cm/sec. In an ocular pharmacokinetic study in the rabbit eye, constant concentrations of each compound were maintained on the cornea in a cylinder or well fixed to the cornea, resulting in a constant input rate. This method circumvented parallel loss routes at the absorption site including nasolacrimal drainage. From area calculations the dispositions of the compounds within the eye were described by mean residence times, steady state volumes of distributions, and clearance rates. R3 and R4 were more slowly absorbed, retained within eye tissues longer, and were cleared more slowly from the eye than ibufenac and ibuprofen. The aqueous humor concentration-time profiles were also computer-fitted to equations representing classical pharmacokinetic models. For ibufenac and ibuprofen, the entire cornea was assumed to be the net barrier for entry into the anterior chamber. Whereas, for R3 and R4, the corneal epithelium and endothelium were presumed to be the diffusional barriers into and out of the stroma, the latter treated as a compartment. Aqueous humor concentrations of each drug fit the models reasonable well and agreed with conclusions made from the use of area calculations. The drop volume method was used to measure the surface tension of each compound. Both ibufenac and ibuprofen were considerably more surface active than R3 or R4. The greater surface tension measured for ibufenac and ibuprofen correlated to the subjective observations of ocular discomfort for these drugs.

Rapid toxicological model for use in assessing ocular drugs.

Nonsteroidal antiinflammatory drugs (NSAIDs) were applied to corneas either by in vitro or in vivo methods. The in vitro method involved excising and mounting corneas in a perfusion system at 37 degrees and exposing drug for 2.5 h. The in vivo methods represent either topical administration to the rabbit eye or topical in vivo infusion using a fixed well which permitted a constant concentration (0.05 per cent) to be applied to the eye of anesthetized rabbits for up to 120 min. An overlay grid procedure using scanning electron microscopy (SEM) showed less per cent endothelial damage with in vivo methods than with the in vitro method of administration, but per cent damage depended on which section was viewed. Damage to the epithelium and endothelium were also assessed by quantitative carboxyfluorescein and Janus green staining and uptake procedures, respectively, following drug exposure by the in vivo infusion method. Results for the epithelium indicated that the more lipophilic NSAIDs damaged the epithelial layer to a greater degree than newly synthesized hydrophilic NSAIDs. Damage to the epithelium correlated to the surface activity of the NSAIDs. Qualitative assessment of epithelial and endothelial toxicity can be performed with SEM and transmission electron microscopy (TEM) while vital staining procedures and the SEM grid procedure can be used to quantitatively assess corneal toxicity. The staining methods, however, possess advantages over SEM and TEM procedures in that they are rapid and do not require laborious preparation. As a result of these characteristics, the vital staining procedures could be used as part of a biopharmaceutical screening technique in evaluating new ophthalmic drugs.

Biopharmaceutical explanation for the topical activity of 6-hydroxyethoxy-2-benzothiazolesulfonamide in the rabbit eye.

6-Hydroxyethoxy-2-benzothiazolesulfonamide (compound 1), 6-hydrogen-2-benzothiazolesulfonamide (compound 2) and ethoxzolamide (compound 3) are carbonic anhydrase inhibitors, of which only compound 1 is active in lowering intraocular pressure when administered to the rabbit eye. They were compared for potency by inhibiting carbonic anhydrase I (CAase I) using the pH stat procedure. No differences were detected. Binding affinity and number of binding sites were identical; only a single binding site was operative. 14C-labelled compound 1 was used to measure the presence of an ocular metabolite which ranged from 17 to 57% in cornea, iris/ciliary body and aqueous humor. By maintaining drug solutions on either the corneal or conjunctival/scleral surfaces of the eye of anesthetized rabbits for 120 minutes, steady state concentrations of compounds 1-3 were determined in cornea, aqueous humor and iris/ciliary body. It was concluded that compound 1 penetrated both routes equally well and also accumulated at the active site in considerably higher concentrations than compounds 2 and 3. Compounds 2 and 3 did not accumulate in the iris/ciliary body nor did compound 3 penetrate the conjunctival/scleral route very well, approximately 5-20 fold lower than from the corneal route.