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Ocular drug delivery offers unique challenges and opportunities in the era of novel therapeutic agents ranging from small molecules to gene therapies. Noninvasive delivery of drugs into the back of the eye or any part of the eye is extremely limited by short precorneal residence time and formidable biological barriers. The eye is a sensitive, sensory organ that requires a high level of material and procedural safety, while achieving therapeutic efficacy. Some recent advances and unmet needs for ocular drug delivery and disposition are discussed in this article. Specifically, nanomedicines, physical and chemical means to enhance delivery, stimuli-responsive delivery systems, the role of vitreal binding on ocular pharmacokinetics, and the influence of aging eye on drug delivery, and the associated unmet needs are highlighted. Additionally, the unmet needs in the medication management for the elderly patients with eye diseases are discussed.

Prostaglandin F2alpha 1-ethanolamide (prostamide F2alpha) is a potent ocular hypotensive agent in animals and represents a new class of fatty acid amide compounds. Accumulated evidence indicated that anandamide, an endogenous bioactive ligand for cannabinoid receptors, may serve as a common substrate to produce all prostamides, including prostamide F2alpha. After incubation of anandamide with cyclooxygenase 2 (COX-2), the reaction mixture was profiled by HPLC and an intermediate metabolite was discovered and characterized as a cyclic endoperoxide ethanolamide using HPLC-tandem mass spectrometry. Formation of prostamide F2alpha was also demonstrated when the intermediate metabolite was isolated and incubated with prostaglandin F synthase (PGF synthase). These results suggest that the biosynthesis of prostamide F2alpha proceeds in two consecutive steps: oxidation of anandamide to form an endoperoxide intermediate by COX-2, and reduction of the endoperoxide intermediate to form prostamide F2alpha by PGF synthase. This endoperoxide ethanolamide intermediate has been proposed as prostamide H2.

Ciclosporin is a potent immunomodulator that acts selectively and locally when administered at the ocular surface. 0.05% ciclosporin ophthalmic emulsion has recently been approved by the US FDA for treatment of keratoconjunctivitis sicca (KCS) [dry-eye disease]. After topical application, ciclosporin accumulates at the ocular surface and cornea, achieving concentrations (/=0.236 microg/g) that are sufficient for immunomodulation. Very little drug penetrates through the ocular surface to intraocular tissues. Ciclosporin is not metabolised in rabbit or dog eyes and may not be prone to metabolism in human eyes. Cultured human corneal endothelial and stromal cells exposed to ciclosporin in vitro exhibited no adverse effects and only minor effects on DNA synthesis. No ocular or systemic toxicity was seen with long-term ocular administration of ciclosporin at concentrations up to 0.4%, given as many as six times daily for 6 months in rabbits and 1 year in dogs. Systemic blood ciclosporin concentration after ocular administration was extremely low or undetectable in rabbits, dogs and humans, obviating concerns about systemic toxicity. In 12-week and 1-year clinical safety studies in dry-eye patients, the most common adverse event associated with the ophthalmic use of ciclosporin emulsion was ocular burning. No serious drug-related adverse events occurred. These data from in vitro, nonclinical and clinical studies indicate effective topical delivery of ciclosporin to desired target tissues along with a favourable safety profile, making 0.05% ciclosporin ophthalmic emulsion a promising treatment for KCS.

To determine aqueous humor concentrations of brimonidine given the following ophthalmic formulations in female New Zealand White Rabbits: (1) BAK-preserved brimonidine tartrate 0.20% at a pH of 6.4; (2) BAK-preserved brimonidine tartrate 0.15% at a pH of 6.4, and (3) Purite((R))-preserved brimonidine tartrate 0.15% at a pH of 7.3.Eighteen (18) animals were given a 35-microL drop of formulation into each eye. Aqueous humor samples were collected at 9 time points over 8 hours. Brimonidine concentrations were quantified using LC-MS/MS.The C(max) was achieved between 0.33-0.67 hours postdosing for all 3 formulations. Mean C(max) after Purite-preserved brimonidine tartrate 0.15% was 88% higher than that after BAK-preserved brimonidine tartrate 0.15% (p = 0.040), and 44% higher than that after BAK-preserved brimonidine tartrate 0.20% (p = 0.0784). AUC(0-3 hr) values were comparable for all 3 formulations.Purite-preserved brimonidine tartrate 0.15% produced higher peak concentrations than BAK-preserved brimonidine tartrate 0.15%. It also had a concentration that was comparable to BAK-preserved brimonidine tartrate 0.20%. The differences in safety may result from the change in preservative.

Tazarotene is an acetylenic retinoid which is metabolised to tazarotenic acid and which binds selectively to the retinoid receptors RARbeta and RARgamma. The safety, toxicity and pharmacokinetics of oral tazarotene were determined over 12 weeks of treatment in 34 patients with advanced cancer. Commonly seen toxicities were mucocutaneous symptoms, musculoskeletal pain and headache. Dose-limiting toxicities were hypercalcaemia, hypertriglyceridaemia and musculoskeletal pain. The maximum tolerated dose of tazarotene in this schedule is 25.2 mg day(-1). Plasma concentrations of tazarotenic acid were found to peak rapidly within 1-3 h of dosing and thereafter declined quickly. The C(max) and AUC values on day 0, and weeks 2 and 4 were similar indicating no drug accumulation. The dose-normalised C(max) and AUC values at different dose levels and different study days appeared to be similar indicating linear pharmacokinetics. No objective responses were seen, although stable disease was seen in six out of eight evaluable patients receiving the three highest dose levels of tazarotene (16.8, 25.2 or 33.4 mg day(-1)). We conclude that oral tazarotene is well tolerated when administered daily for 12 weeks, has a favourable toxicity profile compared with other retinoids and merits further investigation as an anticancer therapy.

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

Purpose. To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies.

To quantify blood cyclosporin A (CsA) concentrations during treatment with CsA topical ophthalmic emulsions, blood was collected from 128 patients enrolled in a Phase 3, multicenter, double-masked, randomized, parallel-group study of CsA eyedrops for treatment of moderate to severe dry eye disease. Patients received 0.05% CsA, 0.1% CsA, or vehicle b.i.d. for 6 months; vehicle-treated patients then crossed over to 0.1% CsA b.i.d. for 6 months. CsA concentrations were measured using a validated LC/MS-MS assay (quantitation limit = 0.1 ng/mL). No patient receiving 0.05% CsA had any quantifiable CsA in the blood (n = 96 samples). All but 7 of 128 (5.5%) trough blood samples from the 0.1% CsA group were below the quantitation limit for CsA; none exceeded 0.3 ng/mL. CsA was also below the limit of quantitation in 205 of 208 (98.6%) of serial postdose blood samples collected from 26 patients during 1 dosing interval between months 9 and 12. The highest C(max) measured, 0.105 ng/mL at 3 hours postdose, occurred in a 0.1% CsA-treated patient. These results indicate that long-term use of topical CsA ophthalmic emulsions at doses that are clinically efficacious for treating dry eye will not cause any system-wide effects.

The objectives of the study were to evaluate the distribution of brimonidine (alpha2-adrenergic agonist) into anterior and posterior ocular tissues. Single or multiple doses of a 0.2 or 0.5% brimonidine tartrate solution were administered to one or both eyes of monkeys or to one eye of rabbits. Brimonidine was administered intraperitoneally to rats. After topical administration, [14C]brimonidine was rapidly absorbed into the cornea and conjunctiva and distributed throughout the eye. [14C]Radioactivity was higher and cleared more slowly in pigmented tissues (iris/ciliary body, choroid/retina, and optic nerve) than in nonpigmented tissues. Single and multiple dosing led to a similar drug distribution, with higher levels of brimonidine measured in pigmented tissues after multiple dosing. Most of the radioactivity extracted from ocular tissues represented unchanged brimonidine. In the rabbits and the monkey treated in only one eye, levels of radioactivity in the untreated eye were low, consistent with the low systemic levels and rapid drug clearance. Posterior ocular tissue concentrations of radioactivity exceeded systemic blood concentrations. The vitreous humor brimonidine concentrations in monkeys treated topically with 0.2% brimonidine tartrate was 82 +/- 45 nM. Vitreous levels in rabbits confirmed the penetration of brimonidine to the posterior segment. Similar concentrations of brimonidine (22 to 390 nM) were measured in the vitreous and retina of rats injected intraperitoneally with brimonidine. Both topically applied and systemically administered brimonidine reach the back of the eye at nanomolar concentrations sufficient to activate alpha2-adrenergic receptors. The brimonidine levels achieved at the retina are relevant for neuroprotection models.

This study compared the precision and accuracy of 4 tear sampling methods. In vivo, albino rabbits were treated with single bilateral eye drops ofofloxacin 0.3% solution, 3 hr after which tear samples were collected using capillary tubes (CT), surgical sponges (SS), or tear strips for 15 sec (15sTS) or 60 sec (60sTS). In vitro, CT, SS, and tear strips were spiked with known volumes of ofloxacin solution in order to assess the bioanalytical accuracy of each technique. Ofloxacin levels were quantified by HPLC in all samples. Results showed that tear volumes and ofloxacin masses sampled in vivo depended on sampling method. Tear volume followed the rank order 60sTS > 15sTS > SS > CT. The volume collected by 60sTS exceeded precorneal tear volume. Ofloxacin mass followed the order 60sTS approximately 15sTS > SS > CT. Tear concentrations (mean +/- SD; N = 12) were 3.28 +/- 3.76 microg/g for 60sTS, 10.3 +/- 10.0 microg/g for 15sTS, 9.75 +/- 8.04 microg/g for SS, and 5.83 +/- 3.35 microg/g for CT. In vitro, SS, 15sTS, and 60sTS yielded accuracies of 103-107% and coefficients of variation (CV) < 9%. CT was only 85% accurate with a CV of 31%, indicating incomplete extraction during analysis. We concluded from this study that: 1) rabbit tear sampling by SS was rapid, easy, accurate, precise, and easily analyzable; 2) sampling by CT or 15sTS was accurate, but may require aggressive extraction (for CT) or be affected by tear flow rate (for 15sTS); and 3) tear sampling by 60sTS underestimated actual tear concentrations.

Tazarotene (AGN 190168) is a new acetylenic retinoid which is effective for the topical treatment of patients with stable plaque psoriasis and mild to moderate acne vulgaris. Topical gel application provides direct delivery of tazarotene into the skin. At 10 hours after a topical application of 0.1% tazarotene gel to the skin of healthy individuals and patients with psoriasis, approximately 4 to 6% of the dose resided in the stratum corneum and 2% of the dose distributed to the viable epidermis and dermis. Tazarotene is rapidly hydrolysed by esterases to its active metabolite, tazarotenic acid. Tazarotenic acid does not accumulate in adipose tissue, but undergoes further metabolism to its sulfoxide and to other polar metabolites and is rapidly eliminated via both urinary and faecal pathways with a terminal half-life of about 18 hours. Percutaneous absorption is similar between healthy individuals and patients with facial acne, leading to plasma concentrations below 1 microg/L. The systemic bioavailability of tazarotene (measured as tazarotenic acid) is low, approximately 1% after single and multiple topical applications to healthy skin. In patients with psoriasis under typical conditions of use, systemic bioavailability increased during the initial 2 weeks of treatment from 1% (single dose) to 5% or less (steady state). The increased bioavailability is probably related to decreases in plaque elevation and scaling due to successful treatment, resulting in a less effective skin penetration barrier to tazarotene. Steady-state concentrations of tazarotenic acid are achieved within 2 weeks of topical treatment in both healthy and psoriatic skin types. The large variability in plasma concentrations observed in patients with psoriasis is probably because of the large differences in lesional skin condition, the amount of drug applied and the surface area of application. There was no significant drug accumulation in the body with long term treatment of patients with psoriasis. Topical administration of tazarotene requires dosages much smaller than those usually required for oral retinoids, such as isotretinoin, acitretin and etretinate, and it delivers the drug directly into the target skin tissues. The low systemic absorption and rapid systemic elimination of tazarotene and tazarotenic acid results in limited systemic exposure. Thus, topical tazarotene has a low potential for systemic adverse effects and is effective in the treatment of patients with acne and psoriasis.

The objective of this study was to assess the corneal and scleral permeabilities of natural prostaglandins as well as their prodrugs and analogs through human cornea and sclera in vitro. The "apparent permeability coefficients" (Papp) of natural prostaglandins (PGF2alpha, PGD2 and PGE2), ester prodrugs of PGF2alpha (1-isopropyl PGF2alpha, 11-pivaloyl PGF2alpha and 11,15-dipivaloyl PGF2alpha) and four analogs (16-m-chlorophenoxy tetranor PGF2alpha, 17-phenyl trinor PGF2alpha, 17-phenyl trinor PGE2 and AH 13205) were measured using modified Ussing perfusion chambers and quantitative high performance liquid chromatography. Our results indicate that the corneal penetration of natural prostaglandins (PGs) is poor (the Papp values ranged from 1.65 x 10(-6) to 2.38 x 10(-6) cm/sec), while the PGF2alpha prodrugs showed higher corneal penetration than PGF2alpha. The 11-pivaloyl ester of PGF2alpha penetrated the cornea faster than both 1-isopropyl ester and the lipophilic 11,15-dipivaloyl ester. The PG analogs also showed poor corneal penetration (Papp values ranged from 0.696 x 10(-6) to 1.49 x 10(-6) cm/sec) except for AH 13205. All compounds tested showed good scleral penetration (Papp values ranged from 6.90 x 10(-6) to 17.1 x 10(-6) cm/sec) except PGF2alpha 11,15-dipivaloyl (Papp = 1.22 x 10(-6) cm/sec). The penetration profiles correlated well with tissue uptake ratios (ratio of final tissue concentration to initial dose) for all compounds except 11,15-dipivalate PGF2alpha. All ester prodrugs (but not the PGs and analogs) underwent corneal first-pass metabolism. The study results demonstrate that transcleral absorption may play a significant role in the ocular absorption of these compounds.

Tazarotene, a member of a novel class of synthetic acetylenic retinoids, is the first topical retinoid to demonstrate both efficacy and an acceptable patient tolerability profile in the treatment of psoriasis. The subchronic and chronic toxicity of topically applied tazarotene gel (0.025-0.1%; 0.05-0.5 mg/kg/d) was evaluated in Hanford miniswine in two separate, prospective, randomized, vehicle-controlled studies of 3 months' and 12 months' duration. Although tazarotenic acid, the active metabolite of tazarotene, was detected in the blood of animals in both studies, no signs of systemic toxicity could be detected in hematology, blood chemistry, posttreatment physical examination, necropsy, or histopathological examination of organ tissues. In both studies, the reversible signs and symptoms of cutaneous irritation caused by topical application of tazarotene gel were consistent with those effects commonly produced by other topical and oral retinoids.

The suitability of a dynamic lung slice culture system as an in vitro model for studying pulmonary metabolism of PGF 2 α was assessed. [ 3 H]Prostaglandin F 2 α ([ 3 H]PGF 2 α ), a twenty carbon fatty acid that contains a five-carbon ring and is known to be metabolized by lung in vivo, was incubated with precision-cut rat lung slices in 1.7 ml of Waymouth's buffer fortified with 10% fetal calf serum. At 0, 2, 4 and 8 h after addition of [ 3 H]PGF 2 α (1.82 ng/μCi), incubation was stopped and the contents of each vial were analyzed for [ 3 H]PGF 2 α and its metabolites using reversed-phase HPLC with radiochemical detection. PGF 2 α was metabolized to 15-keto PGF 2 α , 13,14-dihydro-15-keto PGF 2 α , and two unknown minor polar metabolites. These results indicate that PGF 2 α was metabolized in lung slices pathways similar to those seen in vivo. Slice viability was assessed by protein synthesis and light microscopic examination of lung slices through 24 h of incubation. Protein synthesis was maintained and no tissue necrosis was observed over the entire 24 h incubation, indicating that the lung slices were viable for at least 24 h. These results indicate that the dynamic lung slice culture system is an appropriate in vitro model for studying the pulmonary metabolism of PGF 2 α .

AGN 191103 is an α2-adrenergic agonist that lowers intraocular pressure in animals, but produces dose-dependent sedation. The objective of this study was to test the hypothesis that a 0.2% pH 8.2 ophthalmic AGN 191103 formulation yields comparable ocular concentrations as, but lower systemic concentrations than, a 1% pH 7.2 formulation. Specifically, AGN 191103 concentrations in aqueous humor and plasma were measured for 24 h after acute administration of 14C-AGN 191103 formulated as a 0.94% pH 7.2 or 0.24% pH 8.2 solution and for 8 h after 712 days of twice-daily administration of AGN 191103 formulated as a 0.2% pH 7.2 or 0.2% pH 8.2 solution. After acute administration of the 0.94% pH 7.2 or 0.24% pH 8.2 solution, there was no difference in aqueous humor AUC0−tlast (5879 ± 541 vs. 5697 ± 385 ng·h/ml, respectively; p > 0.5) or Cmax (1310 ± 160 vs. 1630 ± 180 ng/ml, respectively; p = 0.238) in dosed eyes. Plasma AUC0−tlast was dose-proportionally 77% lower (3.37 ± 0.35 vs. 14.8 ± 3.3 ng·h/ml) and Cmax was 75% lower (1.40 ± 0.16 vs. 5.59 ± 2.87 ng/ml) after 0.24% pH 8.2 administration than 0.94% pH 7.2 dosing. The ratio of aqueous humor AUC0−∝ to plasma AUC0−∝ was 333% higher after dosing of 0.24% pH 8.2 than of 0.94% pH 7.2 solution. After repeated twice-daily instillation of 0.2% pH 7.2 or 0.2% pH 8.2 solution, aqueous humor AUC0−tlast after pH 8.2 administration was 304% that following pH 7.2 dosing (1120 ± 140 vs. 368 ± 120 ng·h/ml; p 0.1). The ratio of aqueous humor AUC0−∝ to plasma AUC0−∝ was 339% higher after dosing with 0.2% pH 8.2 than 0.2% pH 7.2 solution. These results indicate that in albino rabbits a 0.2% pH 8.2 AGN 191103 solution produces comparable aqueous humor concentrations as a 1% pH 7.2 formulation, but only one-fourth the plasma concentrations.

We investigated the effect of formulation pH and buffer concentration on the ocular bioavailability of AGN 191103, a basic amine that lowers intraocular pressure. Our objective was to increase AGN 191103's ocular bioavailability enough to allow a clinically significant reduction in dose. Formulations contained 14 C-AGN 191103 (0.1–1% w/v), phosphate or borate buffer (0–30 mM), HCl to pH of 7.4–8.5 and benzalkonium chloride. The first experiment assessed the effect of pH and buffer concentration on the ocular bioavailability of 1% formulations; the second identified conditions of drug and buffer concentration and pH that would yield ocular concentrations comparable to that of a 1%, pH 7.2, 30 mM phosphate formulation known to be effective in vivo. Albino rabbits were given one 35-μl eyedrop, 6 h after which aqueous humors (AqH), corneas and iris-ciliary bodies (ICB) were collected. AGN 191103 is not metabolized by rabbit eyes, so samples were analyzed by liquid scintillation counting with or without combustion. Concentrations in AqH and cornea increased with increasing pH; this trend became more pronounced with increasing buffer concentration. As pH increased from 7.4 to 8.5: (1) corneal concentrations increased 94% and 300%, respectively, after unbuffered and 30 mM buffered formulation administration; (2) AqH concentrations increased 197% and 462%, respectively, after unbuffered and 30 mM buffered formulation administration; and (3) ICB concentrations did not significantly change, or increased 84%, respectively, after ubbuffered or 30 mM buffered formulation administration. Increasing buffer concentration did not affect tissue concentrations at pH 7.4, but significantly increased them at pH 8.5. A 0.2%, pH 8.2, 30 mM borate formulation elicited AqH concentrations comparable to those produced by the 1%, pH 7.2, 30 mM phosphate formulation. Our results indicate that increasing the formulation pH from 7.2 to 8.2 may allow a 5-fold reduction in the dosing concentration, which will markedly reduce the potential for systemic side effects.

The barrier epithelia of the cornea and retina control drug and nutrient access to various compartments of the human eye. While ocular transporters are likely to play a critical role in homeostasis and drug delivery, little is known about their expression, localization and function. In this study, the mRNA expression levels of 445 transporters, metabolic enzymes, transcription factors and nuclear receptors were profiled in five regions of the human eye: cornea, iris, ciliary body, choroid and retina. Through RNA expression profiling and immunohistochemistry, several transporters were identified as putative targets for drug transport in ocular tissues. Our analysis identified SLC22A7 (OAT2), a carrier for the anti-viral drug, acyclovir, in the corneal epithelium, in addition to ABCG2 (BCRP), an important xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment epithelium. Collectively, our results provide an understanding of the transporters that serve to maintain ocular homeostasis and which may be potential targets for drug delivery to deep compartments of the eye.

This study was designed to examine the effect of benzalkonium chloride/ethylenediaminetetraacetic acid (BAK/EDTA) on the ocular bioavailability (Focular) of ketorolac tromethamine after ocular instillation to normal and de-epithelialized corneas of rabbits both in vitro and in vivo. The in vitro Focular of the formulations was measured in flow-through perfusion chambers. For in vivo studies, a 35 microL dose of 0.5% ketorolac tromethamine with or without BAK/EDTA was instilled into rabbit eyes with intact or de-epithelialized corneas. At 0.5, 1, 2, 4, 6, and 8 h postdose, rabbits were euthanized, and the corneas and aqueous humor were collected from both eyes. The ketorolac concentrations from both in vivo and in vitro samples were quantified by reversed-phase high-performance liquid chromatography. The in vitro study results indicated that BAK/EDTA statistically significantly increased the Focular of ketorolac through de-epithelialized corneas but not through intact corneas. The in vivo study results showed that BAK/EDTA had no effect on the Focular of ketorolac in rabbits with intact corneas, based on the values of the area under the aqueous humor concentration versus time curves (AUC0-6h) of ketorolac. As expected, de-epithelialization of the corneas produced a faster and greater ocular absorption of ketorolac as evidenced by the smaller Tmax and larger AUC values compared to those for the intact corneas in vivo. However, BAK/EDTA decreased the ocular absorption of ketorolac in rabbits with de-epithelialized corneas. The half-lives (t 1/2) of ketorolac in corneal tissue and aqueous humor were longer in rabbits with intact corneas than those in rabbits with de-epithelialized corneas. In conclusion, the in vivo Focular of ketorolac was not altered by BAK/EDTA in rabbits with intact corneas, but it was decreased by BAK/EDTA in rabbits with de-epithelialized corneas. Therefore, the formulation with ketorolac alone may be better as a post-operative ocular analgesic.

Idiosyncratic drug reactions (IDRs) that involve the formation of toxic metabolites followed by covalent binding to cellular proteins often go undiscovered until after post-marketing. The goal of this article is to review the current status of IDRs, potential mechanisms and the challenges associated with predicting drug toxicity.The authors review the metabolic pathways of five select classes of sulfur-containing drugs (captopril, troglitazone, tienilic acid, zileuton, methimazole and sudoxicam) suggesting that bioactivation plays a crucial role in the occurrence of IDRs. The reader will gain further awareness that the sulfur atom can propagate as the bioactivation site for the formation of reactive and conceivably toxic metabolites. As such, it is the body's capacity to detoxify these drug products that may determine whether IDRs occur.Incomplete understanding of mechanisms culminating in IDR occurrence represents a monumental impediment toward their abrogation. Moreover, current technology utilized to predict their manifestation (including structure-toxicity relationships) is not infallible and thus, development of novel tools and strategies is indispensible. In an attempt to streamline clinical development and drug approval processes, consortiums have been instated under the US FDA Critical Path Initiative. Collectively, these parameters along with the availability of validated biomarkers and new/updated regulatory guidance could positively influence the outcome of drug toxicity profiles and direct future drug development.

The pharmacokinetics of AGN 190168, a novel synthetic retinoid, and its major metabolite, AGN 190299, in rat blood after intravenous administration was investigated. Approximately 4.4 mg kg-1 (high dose) or 0.49 mg kg-1 (low dose) of AGN 190168 was administered to rats via the femoral vein. Blood was collected from the femoral artery at various time points during an 8 h period. Blood concentrations of AGN 190168 and AGN 190299 were determined by a specific and sensitive high-pressure liquid chromatographic (HPLC) method. AGN 190168 was rapidly metabolized in rats. The only detectable drug-related species in the blood was AGN 190299. Therefore, only pharmacokinetics of AGN 190299 were calculated. Elimination of AGN 190299 appeared to be non-linear after administration of the high dose, and linear after administration of the low dose. The maximum elimination rate (Vmax) and the concentration at half of the Vmax (km), as estimated by a Michaelis-Menten one-compartment model, were 7.58 +/- 2.42 micrograms min-1 (mean +/- SD) and 6.10 +/- 1.58 micrograms mL-1, respectively. The value of the area under the blood concentration time curve (AUC) was 9.54 +/- 1.68 micrograms h mL-1 after administration of the high dose and 0.594 +/- 0.095 micrograms h mL-1 after administration of the low dose. The clearance value was 7.79 +/- 1.20 mL min-1 kg-1 after the high dose, statistically significantly different from that after the low dose (p < 0.05), 14.0 +/- 2.2 mL min-1 kg-1. The terminal half-life (t1/2) was 1.25 +/- 0.74 h for the high-dose group and 0.95 +/- 0.16 h for the low-dose group. Study results demonstrate rapid systemic metabolism of AGN 190168 to AGN 190299, non-linear pharmacokinetics of AGN 190299 after the 4.4 mg kg-1 dose, and the lack of difference in disposition profiles between sexes after intravenous administration of AGN 190168 to rats.

The pharmacokinetic profiles of 0.3% ofloxacin and 0.3% tobramycin ophthalmic solutions after multiple administrations in the eyes of 160 healthy volunteers were evaluated. In human tears, ofloxacin and tobramycin were found to have terminal half-lives of 226 and 154 minutes, respectively. The mean residence time in the ocular tear fluid was 326 minutes for ofloxacin and 106 minutes for tobramycin. The mean duration of time that ofloxacin remained above the MIC90 value for five bacterial species evaluated was 605 minutes, compared with 251 minutes for tobramycin. The mean area under the inhibitory curve for the five bacterial species evaluated was greater for ofloxacin (2224) compared with tobramycin (1549). The duration of time above the MIC90 for ofloxacin was longer compared with tobramycin for gram-positive isolates. Overall, the pharmacokinetic and pharmacodynamic profiles of ofloxacin were superior to those of tobramycin for most parameters studied.

Studies in vitro and in vivo were conducted to investigate the effect of pilocarpine on ocular absorption of levobunolol when both drugs were formulated in one solution dosage. The ocular absorption of levobunolol is pH-dependent. Due to the large buffering capacity of pilocarpine at pH 5.5, the ocular absorption of levobunolol from pilocarpine-containing solutions was reduced by approximately four-fold as compared to a non-pilocarpine-containing formulation at pH 7.2. The ocular absorption of levobunolol in the presence of pilocarpine at acidic pH was enhanced by the use of sulfosuccinates, specifically Schercopol CMS.

We read with great interest the recent article by Ogundele and Jasek,1 in which the authors concluded that bimatoprost ophthalmic solution 0.01% (Lumigan®; Allergan, Inc, Irvine, CA) produced lower bimatoprost acid concentration than bimatoprost ophthalmic solution 0.03% (Lumigan; Allergan, Inc) in the aqueous humor of rabbits. This conclusion was made based on two treatment time points (30 and 90 minutes) with a small sample size (n = 4) at each time point and with large variability. In comparing pharmacokinetic profiles of two formulations, it is a general practice that the study design would support assessment of the speed of onset (ie, time to maximum exposure [Tmax]), and the extent of absorption (ie, maximum concentration and area under the concentration-time curve). Therefore, a more complete temporal profile would be necessary. An erroneous conclusion could be drawn based on two seemingly arbitrary time points. For example, it is unclear whether these two time points reside in the ascending or descending portion of the temporal profile or if they reside one in each portion and the Tmax is missing. Based on two data points, Ogundele and Jasek1 hypothesized that bimatoprost 0.01% might have compromised the intraocular pressure (IOP)-lowering effect. Not only is there no reported correlation of animal pharmacokinetic and/or metabolism results to clinical efficacy, this hypothesis directly contradicts existing evidence. A multicenter, 12-month, randomized, controlled trial demonstrated equivalent efficacy of bimatoprost 0.01% and bimatoprost 0.03% based on predetermined IOP criteria (limits of the 95% confidence interval of the between-group difference in mean IOP within ±1.5 mmHg at all time points and within ±1 mmHg at most time points).2 In addition, Ogundele and Jasek1 speculated that bimatoprost 0.01% might have increased the risk of ocular toxicity, based on prior publications of in vitro and animal studies that may not be relevant to patients, case series, or open-label clinical studies. This evidence is considered lower level compared with randomized, controlled trials. Katz et al2 conducted a randomized, controlled trial and reported bimatoprost 0.01% had (a) significantly lower overall incidence of treatment-related adverse events (P < 0.03); (b) significantly reduced conjunctival hyperemia (P < 0.044), skin pigmentation (P < 0.02), and eye pruritus (P < 0.035); and (c) significantly lower discontinuation rates than bimatoprost 0.03% (P = 0.043). Based on clinical trial evidence, the claims of ocular toxicity are unfounded for at least 12 months of administration as monotherapy.2 In addition, a recent clinical study comparing ocular surface tolerability of topical prostaglandin analogs demonstrated no statistical differences following 3 months of treatment in either corneal staining or conjunctival hyperemia between latanoprost, which has 200 ppm benzalkonium chloride (BAK), similar to bimatoprost 0.01%, and travoprost ophthalmic solution 0.004% (Travatan Z®; Alcon Laboratories, Inc, Fort Worth, TX) (BAK free, preserved with sofZia® [Alcon Laboratories, Inc]).3 Bimatoprost 0.01%, which offers improved ocular tolerability while maintaining the established efficacy of bimatoprost 0.03%, recently gained approval by the European Medicines Evaluation Agency (2009) and the US Food and Drug Administration (2010) as first-line therapy in patients with open-angle glaucoma or ocular hypertension. Any ophthalmologist or patient concerns raised by the incomplete and potentially misleading conclusion published by Ogundele and Jasek1 can be addressed with a carefully constructed pharmacokinetic study with a complete time-concentration profile. We are currently preparing a manuscript that reports the findings of such a study design.

The objective of the study was to investigate the systemic disposition of 14C-SK&F L-190144 after single intravenous (10mg kg−1) and oral (200 mg kg−1) doses to rats and after single intravenous and ocular doses (0.33 mg kg−1) to monkeys. After the intravenous dose, the blood concentration-time profile of 14C-SK&F L-190144 followed a rapid triexponential decline with half-lives of 2.5, 15, and 246 min in rats and 3, 19, and 2520 min in monkeys. The 14C-label in blood was mainly the parent compound. The terminal elimination half-life detected in rats using the urinary excretion rate-time data was 700 min. The total body clearance values were 17.6 ± 2.1 (mean ± SD, n = 6) and 1.11 ± 0.41 (n=4) mlmin−1 kg−1 for rats and monkeys, respectively. Both species had similar values of volume of distribution at the terminal phase, 4 to 6 l kg−1, and similar excretion patterns, approximately 60 per cent and 30 per cent of the dose were excreted in the urine and feces, respectively. 14C-SK&F L-190144 was not absorbed orally in rats with the majority of the dose recovered in the feces. Following ocular administration to monkeys, the plasma drug concentrations peaked at 8 h post-dosing but did not reach a biexponential elimination phase until 18 h post-dosing, suggesting slow systemic absorption of drug from the ocular site. The monkeys excreted 42 per cent of the dose in urine and 50 per cent in feces after ocular administration. This increase in fecal excretion compared to the intravenous route of administration may have been due to the slow absorption by the ocular and nasal tissues altering the relative proportions of drug elimination via the renal and hepatic routes, or to a proportion of the dose passing into the gastrointestinal tract and exiting unabsorbed. Study results demonstrate similar excretion patterns and volume of distribution after intravenous administration in both species. The slow terminal elimination phase in monkeys was attributed to the low body clearance. The low oral bioavailability was possibly due to the poor partitioning behavior of the drug (logarithm of partition coefficient - 2.6). A significant fraction of the dose was absorbed in the body via the ocular route.

An in vitro method for investigating drug penetration across the cornea from acute doses has been developed. The donor (epithelial or tear) side of a corneal chamber similar to those used by Edelhauser and co-workers1, 2 was modified with a reduced-volume insert so that the donor side volume approximated 6% of the receiver (endothelial or aqueous humor) chamber volume. Fluid was pumped through the anterior chamber to simulate the physiologic tear turnover in vivo. The receiver chamber was bubbled with oxygen:carbon dioxide (95%:5%) to aerate and circulate the fluid. We investigated the in vitro ocular bioavailability of several marketed ophthalmic drug formulations using this model (0.03% flurbiprofen, 0.5% levobunolol, 0.1 and 0.25% fluorometholone, and 1% prednisolone acetate). At 1 min after the dose administration, drug was eluted from the donor chamber at a dose turnover rate of 12%/min. In all experiments, a small percentage of the applied dose penetrated the cornea. Using this chamber device, the ocular bioavailability via corneal absorption ranged between 5 and 16% for solution formulations, whereas the ocular bioavailability for the suspension formulations was

PURPOSE. Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. METHODS. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naive untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1 % or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [ 3 H]testosterone. RESULTS. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naive rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naive rabbit lacrimal gland at a rate of 14 ± 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [ 3 H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [ 3 H]testosterone 6β hydroxylation in a concentration-dependent manner, with IC 50 ranging from 3.73 to 435 μM. CONCLUSIONS. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly prescribed glucocorticoid and can be inhibited by known CYP3A inhibitors.

Ophthalmic drugs typically achieve < 10% ocular bioavailability. A drug applied to the surface of the eye may cross ocular-blood barriers where it may encounter metabolising enzymes and cellular transporters before it distributes to the site of action. Characterisation of ocular enzyme systems and cellular transporters and their respective substrate selectivity have provided new insight into the roles these proteins may play in ocular drug delivery and distribution. Altered metabolism and transport have been proposed to contribute to a number of ocular disease processes including inflammation, glaucoma, cataract, dry eye and neurodegeneration. As ocular enzyme and transport systems are better characterised, their properties become an integral consideration in drug design and development.

Oral tazarotene, an acetylenic retinoid, is in clinical development for the treatment of psoriasis. The disposition and biotransformation of tazarotene were investigated in six healthy male volunteers, following a single oral administration of a 6 mg (100 µCi) dose of [ 14 C]tazarotene, in a gelatin capsule. Blood levels of radioactivity peaked 2 h postdose and then rapidly declined. Total recovery of radioactivity was 89.2 ± 8.0% of the administered dose, with 26.1 ± 4.2% in urine and 63.0 ± 7.0% in feces, within 7 days of dosing. Only tazarotenic acid, the principle active metabolite formed via esterase hydrolysis of tazarotene, was detected in blood. One major urinary oxidative metabolite, tazarotenic acid sulfoxide, accounted for 19.2 ± 3.0% of the dose. The majority of radioactivity recovered in the feces was attributed to tazarotenic acid representing 46.9 ± 9.9% of the dose and only 5.82 ± 3.84% of dose was excreted as unchanged tazarotene. Thus following oral administration, tazarotene was rapidly absorbed and underwent extensive hydrolysis to tazarotenic acid, the major circulating species in the blood that was then excreted unchanged in feces. A smaller fraction of tazarotenic acid was further metabolized to an inactive sulfoxide that was excreted in the urine. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies.TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [14C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (Papp). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075.TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between Papp and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1.TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.

The diffusion from the site of intramuscular injection of 900 kDa botulinum neurotoxin-hemagglutinin complex (BoNT/A-complex) and 150 kDa free-botulinum neurotoxin (free-BoNT/A) was compared. Radioiodinated compounds were injected into the gastrocnemius muscle of rats (70Units (U) 125I-BoNT/A-complex, 67 or 344 U free-125I-BoNT/A, or free-125I-iodide) and the eyelids of rabbits (24 U 125I-BoNT/A-complex or 108 U free-125I-BoNT/A), and measured in various tissues at different time points. There were no detectable systemic effects or generalized botulinum neurotoxin toxicity in either rats or rabbits, indicating that most of the toxin, whether as 125I-BoNT/A-complex or free-125I-BoNT/A, remained at the injection site. In rats, 125I-BoNT/A-complex and free-125I-BoNT/A diffused in a pattern that was grossly similar. Almost no radioactivity was recovered from the brain. Radioactivity recovered from distant tissues (thyroid, skin, and contralateral muscle) was primarily attributable to either low molecular weight 125I-containing peptides or 125I-iodide. After injection into rabbit eyelids, neither 125I-BoNT/A-complex nor free-125I-BoNT/A spread to distant structures, including the eye. The results indicate that most of the neurotoxin does not diffuse from the injection site, whether in free or complexed form, and this may reduce the potential for systemic effects.

Objective: To evaluate the safety and pharmacokinetics of tazarotene cream 0.1% under standard (face only) or exaggerated (15% body surface area, including the face) application conditions after a single dose and after repeat topical applications once daily to patients with acne vulgaris or photodamaged skin. Methods: Two separate, randomised, single-centre, nonblinded, parallel-group pharmacokinetic studies were conducted. In one study, tazarotene cream 0.1% was applied either to the face of eight female patients with moderate acne or to 15% body surface area of ten female patients with severe acne. In the other study, tazarotene cream 0.1% was applied either to the face (six females, two males) or to 15% body surface area (8 females, 8 males) of patients with photodamaged skin. In both studies, tazarotene cream 0.1% was applied once daily (except on days 1 and 2) for 30 days. Blood was drawn for measurement of plasma concentrations of tazarotenic acid at defined time intervals after application of the cream. Plasma tazarotenic acid concentrations were determined by a validated gas chromatography-tandem mass spectrometry method with a lower limit of quantification of 0.005 μg/L. Results: At exaggerated application rates in patients with acne vulgaris, the maximum average peak concentration (Cmax) and 24-hour area under the concentration-time curve (AUC) values of tazarotenic acid were (mean ± SD) 1.20 ± 0.41 μg/L (n = 10) and 17.0 ± 6.1 μg · h/L (n = 10), respectively, and occurred on day 15. The single highest Cmax was 1.91 μg/L. At standard application rates in patients with acne vulgaris, the maximum average Cmax and AUC values of tazarotenic acid were 0.10 ± 0.06 μg/L (n = 8) and 1.54 ± 1.01 μg · h/L (n = 8), respectively, and occurred on day 15. At exaggerated application rates in patients with photodamaged skin, the maximum average Cmax and AUC values of tazarotenic acid were (mean ± SD) 1.75 ± 0.53 μg/L (n = 16) and 23.8 ± 7.0 μg · h/L (n = 16), respectively, and occurred on day 22. The single highest Cmax was 3.43 μg/L on day 29. At standard application rates in patients with photodamaged skin, the maximum average Cmax and AUC values of tazarotenic acid were 0.236 ± 0.255 μg/L (n = 8) and 2.44 ± 1.38 μg · h/L (n = 8), respectively, and occurred on day 15. Gender had no influence on the systemic exposure of tazarotenic acid. The most common treatment-related adverse events were signs and symptoms of local irritation, of mild or moderate severity. Conclusion: The pharmacokinetics of tazarotene cream 0.1% in patients with acne vulgaris or photodamaged skin are similar. The maximum average plasma concentrations of tazarotenic acid after topical application of tazarotene cream 0.1% to the face were less than 0.25 μg/L. The maximum average plasma concentrations of tazarotenic acid following application to an exaggerated body surface area (15%) were less than 1.8 μg/L.

Purite (stabilized oxychloro complex) and benzalkonium chloride (BAK) are preservatives. We investigated formulation effects on ocular absorption of brimonidine in rabbit eyes. The formulations compared were: Alphagan (0.2% brimonidine tartrate/0.005% BAK, pH 6.4), Brimonidine-Purite (0.2% brimonidine tartrate/0.005% Purite, pH 7.2), and Brimonidine-PF (0.2% brimonidine tartrate, preservative-free (PF), pH 6.4) solutions. The study was conducted in a cross-over fashion; albino rabbits (n = 18) were given a single 35 microl drop of each test formulation in each eye. Aqueous humor samples were collected at selected times post-dose from subgroups of 2 rabbits per timepoint and analyzed for brimonidine concentrations by LC-MS/MS. The AUC and Cmax were calculated. The results showed rapid ocular absorption of brimonidine, with peak concentrations at 0.33-1 hr. The AUC(0-5hr) values were 3.78 +/- 0.38, 2.77 +/- 0.22, and 2.49 +/- 0.22 microg-hr/ml (mean +/- SEM) for Brimonidine-Purite, Alphagan and Brimonidine-PF, respectively. The corresponding Cmax values were 2.69 +/- 0.72, 1.74 +/- 0.13, and 1.24 +/- 0.22 microg/ml (mean +/- SEM). Brimonidine-Purite provided significantly higher AUC(0-5hr) than Alphagan (p < 0.05). No statistical significant difference in AUC(0-5hr) was found between Alphagan and Brimonidine-PF. In conclusion, 0.2% Brimonidine-Purite was 1.4 and 1.5 times more ocularly bioavailable in rabbits than 0.2% Alphagan and 0.2% Brimonidine-PF, respectively.

A 1-month-old infant with Peters anomaly had recurrent episodes of unresponsiveness, hypotension, hypotonia, hypothermia, and bradycardia. An extensive medical evaluation determined these episodes to be caused by brimonidine, an anti-glaucoma agent. There is the potential for serious toxic effects from the systemic absorption of topically applied ophthalmic agents in children.

Concentrations of three anti-infective agents in tear film were monitored after one topical application in rabbits. Ofloxacin concentrations exceeded the MIC for 90% of the organisms tested (MIC90) (gram-negative and gram-positive organisms) for 240 min. Tobramycin concentrations exceeded the MIC90 for 10 min. Gentamicin concentrations exceeded the MIC90 for 20 min for gram-positive organisms and 120 min for gram-negative organisms.

Cyclosporine is an immune modulator that inhibits T-lymphocyte-mediated immunoreactivity. Allergan is currently evaluating the clinical efficacy of 0.05%–0.4% cyclosporine emulsion for the treatment of immuno-inflammatory eye diseases, such as keratoconjunctivitis sicca, or dry eye syndrome. Topical ocular application of cyclosporine, formulated as 2% cyclosporine in olive oil, 0.2% cyclosporine in corn oil ointment (Schering-Plough), or 0.2% cyclosporine emulsion (Allergan), was found to reduce ocular surface inflammation and improve lacrimal gland secretion in dogs with KCS.1–3

The objective of this study was to identify prostaglandin F2alpha (PGF2alpha) prodrugs that have an optimal ocular absorption profile and therefore could be potentially useful for the treatment of glaucoma. Rabbit cornea, conjunctiva, and iris/ciliary body were mounted in a flow-through chamber to evaluate the permeability and bioconversion of PGF2alpha and its prodrugs. The prodrugs tested were PGF2alpha 1-isopropyl, 1,11-lactone, 15-acetyl, 15-pivaloyl, 15-valeryl, and 11,15-dipivaloyl esters. After 4 h in the donor or acceptor compartments, the products and formation of PGF2alpha were analyzed by HPLC. Effects on intraocular pressure and ocular surface hyperemia were also determined. All prodrugs penetrated the rabbit cornea faster than PGF2alpha by 4- to 83-fold. All prodrugs penetrated conjunctiva faster than PGF2alpha, except the 15-acetyl ester prodrug, which was equally permeable. No direct correlation between drug lipophilicity and permeability across the cornea or conjunctiva was apparent. The most metabolically stable prodrug was the 1,11-lactone, followed by the 11,15-dipivaloyl, 15-pivaloyl, 15-acetyl, 1-isopropyl, and the 15-valeryl esters, the latter of which was extensively converted to PGF2alpha. A separation index for various prodrugs was calculated from the ratio of the bioavailable PGF2alpha for ocular hypotension to the bioavailable PGF2alpha for hyperemia. The highest separation index was observed for the 1,11-lactone prodrug (2.33), followed by the 11,15-dipivaloyl ester prodrug (1.80). Thus the 1,11-lactone and 11,15-dipivaloyl ester prodrugs appeared to be superior to the others in providing bioavailable PGF2alpha for ocular hypotension, while minimizing hyperemia. The favorable separation index for these compounds appeared to be due to their metabolic stability at the corneal surface and conjunctiva combined with sufficient bioavailability for ocular hypotension.

Tazarotene is a novel acetylenic retinoid for the treatment of psoriasis and acne. We examined (1) the hydrolysis of tazarotene in blood from Japanese-American and Caucasian subjects, (2) the esterases responsible for this hydrolysis in human blood, and (3) tazarotene hydrolysis in rat and human liver microsomes. Tazarotene hydrolysis and enzyme inhibition were assessed by monitoring the disappearance of tazarotene and the appearance of its primary metabolite tazarotenic acid by HPLC. In blood, tazarotene was converted mainly to tazarotenic acid via first-order kinetics, and there was no statistically significant difference in the hydrolytic (metabolic) rate of tazarotene in uninhibited Japanese-American and Caucasian blood. Physostigmine (a cholinesterase inhibitor), bis(p-nitrophenyl) phosphate (a carboxylesterase inhibitor), and EDTA (an aromatic esterase inhibitor) did not significantly affect tazarotene hydrolysis in blood. Paraoxon, an inhibitor of all serine esterases including cholinesterase and carboxylesterase, decreased the hydrolysis of tazarotene to tazarotenic acid by 95% in both blood and liver microsomes. In conclusion, blood and liver esterases play a significant role in the hydrolysis of tazarotene to tazarotenic acid, and paraoxon-inhibitable forms of esterases are involved in this hydrolysis in humans.

1. In vitro metabolism of 14C-brimonidine by the rat, rabbit, dog, monkey and human liver fractions was studied to assess any species differences. In vitro metabolism with rabbit liver aldehyde oxidase and human liver slices, and in vivo metabolism in rats were also investigated. The hepatic and urinary metabolites were characterized by liquid chromatography and mass spectrometry. 2. Up to seven, six, 11 and 14 metabolites were detected in rat liver S9 fraction, human liver S9 fraction, human liver slices and rat urine respectively. Rabbit liver aldehyde oxidase catalysed the metabolism of brimonidine to 2-oxobrimonidine and 3-oxobrimonidine, and further oxidation to the 2,3-dioxobrimonidine. Menadione inhibited the liver aldehyde oxidase-mediated oxidation. 3. Hepatic oxidation of brimonidine to 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine was a major pathway in all the species studied, except the dog whose prominent metabolites were 4',5'-dehydrobrimonidine and 5-bromo-6-guanidinoquinoxaline. 4. These results indicate extensive hepatic metabolism of brimonidine and provide evidence for aldehyde oxidase involvement in brimonidine metabolism. The species differences in hepatic brimonidine metabolism are likely related to the low activity of dog liver aldehyde oxidase. The principal metabolic pathways of brimonidine are alpha(N)-oxidation to the 2,3-dioxobrimonidine, and oxidative cleavage of the imidazoline ring to 5-bromo-6-guanidinoquinoxaline.

Brimonidine is a selective α2-adrenergic agonist developed for lowering intraocular pressure in glaucoma patients. Since brimonidine will be used in long-term theraphy, the safety of this drug is an important feature for its clinical success. Brimonidine has been evaluated in a number of safety studies using doses much greater than those in humans. In this paper chronic and carcinogenicity studies are presented. The results of the 6-month ocular/systemic study in rabbits and the 1-year ocular/systemic study in monkeys with 0.2, 0.5, and 0.8% brimonidine ophthalmic formulations showed no ocular or organ toxicity. The highest concentration of 0.8% used in rabbits and monkeys resulted in plasma drug concentrations of 95 (Cmax) and 10 (C2hr) times, respectively, higher than those seen in humans following topical dosing. Dose-related transient exaggerated pharmacologic effects of sedation were observed in the 1-year oral study in monkeys without any organ toxicity. The dose that elicited an apparent pharmacologic effect produced a plasma drug concentration that was approximately 115 times higher than that in humans. In 2-year carcinogenicity studies in mice and rats using doses that produced plasma concentrations 77 and 118 times, respectively, higher than those seen in humans, no oncogenic effect was observed. Based on the extensive safety research on brimonidine, it was concluded that this drug has an excellent safety profile.

Brimonidine is an alpha 2-adrenergic agonist that is efficacious in lowering intraocular pressure in humans. A highly sensitive and selective gas chromatography/mass spectrometry (GC/MS) assay is described for quantitation of brimonidine in human plasma following ocular installation. Brimonidine in 1 ml of plasma was extracted together with tetradeuterated brimonidine (internal standard) and clonidine (carrier) by solvent extraction. After solvent evaporation, 3,5-bis(trifluoromethyl)benzoyl derivatives were formed and injected onto a GC/MS apparatus under negative chemical ionization conditions. The ions monitored for derivatized brimonidine and tetradeuterated brimonidine were m/z 691 [M-HBr] and m/z 694 [M-DBr], respectively. Calibration curves were linear from 2 to 1000 pg ml-1 (r2 = 0.981-0.996). The method was specific for brimonidine relative to endogenous compounds in plasma. The inter-day relative standard deviation for analysis of quality controls was 12% or less, and the inter-day assay accuracy ranged from 97 to 104% of nominals. The GC/MS assay showed adequate sensitivity for analysis of human samples from volunteers ocularly dosed with 0.5% brimonidine tartrate solution. Overall, the GC/MS assay showed excellent precision and accuracy, and a minimum quantifiable concentration of 2 pg ml-1.

Ofloxacin 0.3% ophthalmic solution or ointment was administered preoperatively to 13 patients undergoing cataract surgery. Mean drug concentrations in conjunctival biopsies were 2.62 and 6.55 micrograms/gm and in aqueous humor samples were 0.36 micrograms/mL and 0.43 micrograms/mL, for ointment and solution respectively. Mean conjunctival concentrations of ofloxacin achieved MIC90 values for 419 gram-positive and gram-negative organisms previously analyzed.

The potential effects of anti-glaucoma drugs, such as levobunolol and timolol, on blood flow in the posterior segment of the eye are of great interest in terms of changes in optic nerve head perfusion and prevention of visual field loss. These effects are related to the rate and extent of their absorption into the site of action. In this study, the concentrations of timolol and levobunolol in the aqueous humor, iris-ciliary body, vitreous humor, choroid-retina, and optic nerve were compared following instillation of a single drop of 0.5% ophthalmic solutions into albino rabbit eyes. Tissue drug and metabolite concentrations were measured by liquid chromatography-mass spectrometry. Dihydrobunolol (DHB) is an equipotent metabolite of levobunolol. In the anterior segment of the eye, levobunolol plus DHB concentrations were higher than timolol concentrations in aqueous humor and were comparable to those of timolol in iris-ciliary body. However, in the choroid-retina and optic nerve, timolol concentrations were greater than those of levobunolol plus DHB. Overall, the study demonstrates comparable concentrations of levobunolol and timolol in the anterior section of the eye. The low availability of levobunolol in the posterior segment as compared to timolol may be a key advantage for levobunolol in producing less adverse effect on blood flow in the choroid-retina and optic nerve.

The usefulness of penetration enhancers in promoting drug permeation across the cornea was investigated for drugs varying from hydrophilic to lipophilic. Four purported penetration enhancers [Azone (laurocapram), hexamethylenelauramide, hexamethyleneoctanamide, and decylmethylsulfoxide] were employed. Corneal permeability coefficients of drugs that were either hydrophilic (acetazolamide, cimetidine, guanethidine, and sulfacetamide), moderately lipophilic (bunolol and prednisolone), or lipophilic (flurbiprofen and its amide analogue) were measured in the absence or in the presence of various Azone concentrations. The effects of penetration enhancers on the corneal penetration of cimetidine were also compared. The corneal penetration of hydrophilic compounds was enhanced by at least 20-fold at 0.1% Azone. For prednisolone and bunolol, the maximal enhancement was at 0.025-0.1% Azone and was marginal (two- to 5-fold), whereas Azone inhibited rather than enhanced the corneal penetration of the lipophilic flurbiprofen and its amide analogue. All four enhancers behaved similarly in enhancing corneal penetration of cimetidine and corneal hydration after incubation in vitro. Possible mechanisms of penetration enhancers on corneal drug penetration were discussed. Penetration enhancers may have clinical benefits in improving ocular drug delivery of hydrophilic compounds, however, their utility may depend on the toxicological profiles.

We evaluated the systemic absorption of ofloxacin eyedrops in humans and their availability in the tear film. Serum, urine, and tear film concentrations of ofloxacin were measured in 30 healthy women topically treated with 0.3% ofloxacin, in both eyes, four times daily for 10 1/2 days. Serum was collected before the first daily dose on days 1 and 11 and at 18 time points before the second dose. Maximum serum ofloxacin concentrations (1.89 +/- 1.13 ng/ml) after 10 1/2 days of topical dosing were more than 1,000 times lower than those reported after standard oral doses of 300 mg ofloxacin. Urine was collected for the 24-h period after the first daily dose on days 1 and 10. Topical ofloxacin was excreted in the urine primarily in unmodified form and recovery rates were significantly higher on day 10 (76.1 +/- 41.5%) than on day 1 (56.6 +/- 31.6%) (p less than 0.05). Both serum and urine data give evidence to accumulations of ofloxacin over a 10 1/2-day period. The low serum concentration at steady state suggests an extremely low potential for producing systemic effects. No systemic side effects attributable to topical ofloxacin were observed. Mild ocular irritation was reported by two patients while under treatment. Tears were collected 4 h after the first treatment on day 11 and at 5, 10, 20, 30, and 40 min after the second treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Ofloxacin, a new quinolone antibiotic with a broad spectrum of activity, is very effective against Pseudomonas aeruginosa in vitro. Its effectiveness was studied in a rabbit model of tobramycin-sensitive P. aeruginosa. Treatment groups received either vehicle, tobramycin 0.3%, or ofloxacin 0.3%. Twelve hours of treatment decreased the bacterial counts from a mean of 2.2 +/- 0.7 x 10(6) colony forming units (cfu) per cornea in the vehicle group to means of 513 +/- 670 and 435 +/- 524 cfu in the tobramycin and ofloxacin groups, respectively. This decrease in bacterial counts was statistically significant (p = 0.001 for tobramycin and ofloxacin each compared with control, p = 0.86 for tobramycin compared with ofloxacin). After seven days, all antibiotic-treated corneas were sterile and the epithelial defects healed at comparable rates. Aqueous humor drug levels were higher in infected eyes without an intact epithelium (p = 0.02); in eyes with intact epithelium, concentrations of ofloxacin were higher than were those of tobramycin (p = 0.002). In this animal model, ofloxacin proved to be an effective antibiotic with no evidence of toxicity.

The objective of this study was to explore the relationship between lipophilicity and the lenticular uptake of radiolabeled xenobiotics. The lenticular uptake of amino acids was also investigated. An organ-culture technique was employed and the partitioning of compounds into the rabbit lens was measured for compounds with log octanol/water partition coefficients (log P o/w) ranging from -1 to 7.3. Drug distribution in the lens was expressed as the concentration ratio of that in the lenticular section (capsule/epithelium, cortex, and nucleus) to that in the incubation medium, (C(lens) section/Cm). The drug partitioning into the lens capsule was facile for all of the compounds tested. The drug concentrations in the lens capsule were higher than those in the cortical and nuclear regions. For compounds with low partition coefficients (such as sulfacetamide and cimetidine), an apparent distribution equilibrium was achieved during a 5-hr period. The C(lens)/Cm value of polar compounds, being less than one, did not further increase by prolonging the incubation period. Only compounds with log Po/w values between 3 and 6 had Cnucleus/Cm values exceeding unity. The maximal values of Ccortex/Cm and Cnucleus/Cm, approximately 15 and 4, respectively, for anthracene (log Po/w = 4.5) and diethylstilbestrol (log Po/w = 5.1), were observed in this study. A bell-shaped relationship was observed between the lenticular uptake rate and drug lipophilicity, of which the maximum occurred around log Po/w of 4. These results indicate the existence of a lipophilicity window for favorable drug distribution into the deeper region of the lens. For several lipophilic compounds, such as padimate-O, values of Cnucleus/Cm increased steadily over a 24-hour incubation period. This suggests that the nucleus behaved as a deep compartment for these compounds. L-Cysteine and L-serine were actively taken up by the lens and the lenticular absorption of L-cysteine was concentration-dependent with Vmax and Km values of 18.3 mumol/gm/hr and 49.8 mM, respectively. In summary, a relationship between lenticular uptake and drug lipophilicity was demonstrated. The optimal log Po/w value for drug uptake into the lens was between 4 and 5. The slowness of reaching significant drug concentrations in the nucleus necessitates a chronic dosing regimen to deliver therapeutic drug levels inside the lens.