Gene expression profiling of transporters in the solute carrier and ATP-binding cassette superfamilies in human eye substructures.

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 antiviral 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.

Idiosyncratic reactions and metabolism of sulfur-containing drugs.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

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 naïve 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 [3H]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 naïve 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 naïve 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 [3H]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 [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. 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.

Disposition and biotransformation of the acetylenic retinoid tazarotene in humans.

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 microCi) dose of [14C]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.

Enzymatic formation of prostamide F2alpha from anandamide involves a newly identified intermediate metabolite, prostamide H2.

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.

Ocular pharmacokinetics and safety of ciclosporin, a novel topical treatment for dry eye.

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.

Tazarotene does not affect the pharmacokinetics and efficacy of a norethindrone/ethinylestradiol oral contraceptive.

OBJECTIVE: To determine the pharmacokinetic and pharmacodynamic interaction between oral tazarotene and an oral contraceptive containing norethindrone 1mg and ethinylestradiol 0.035 mg (Ortho-Novum 1/35). DESIGN: Two separate open-label, parallel-group, single-centre, pharmacokinetic and pharmacodynamic interaction studies. PARTICIPANTS AND METHODS: Twenty-seven healthy women (age 20-55 years) completed Study I, with a duration of 64 days during three consecutive menstrual cycles. Ortho-Novum 1/35 was taken once daily from study day 0 (first cycle day 1) to day 61 (third cycle day 6), and oral tazarotene 1.1 mg was coadministered daily from study day 34 (second cycle day 7) to day 61. Twenty-nine healthy women (age 20-44 years) completed Study II, with a duration of 75 days during three consecutive menstrual cycles. Ortho-Novum 1/35 was taken once daily from study day 0 (first cycle day 1) to day 74 (third cycle day 19), and oral tazarotene 6 mg was coadministered daily from study day 48 (second cycle day 21) to day 74. In both studies, the pharmacokinetics of tazarotenic acid on study day 61 (third cycle day 6) were evaluated from plasma tazarotenic acid concentrations. Pharmacokinetic parameters of plasma norethindrone and ethinylestradiol were compared before and after tazarotene administration (cycle day 6 of the second and third cycles, respectively). Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) concentrations were compared before and after tazarotene administration (cycle days 2, 4 and 6 of the second and third cycles, respectively). In Study II, serum progesterone concentrations were also determined on cycle days 18 and 20 of the second and third cycles. Tazarotenic acid was determined by liquid chromatography-tandem mass spectrometry. Ethinylestradiol and norethindrone were determined by gas chromatography-mass spectrometry. LH and FSH were assayed by microparticle enzyme immunoassay in Study I and by double-antibody radioimmunoassay in Study II. Progesterone was determined by solid-phase radioimmunoassay. RESULTS: In Study I (tazarotene 1.1 mg), the area under the plasma concentration-time curve from zero to 24 hours (AUC24) and the peak concentration in plasma (Cmax) for tazarotenic acid were 121 +/- 27 microg. h/L and 28.9 +/- 9.4 microg/L (mean +/- SD), respectively. In Study II (tazarotene 6 mg), AUC24 and Cmax for tazarotenic acid were 379 +/- 78 microg. h/L and 111 +/- 37 microg/L (mean +/- SD), respectively. In both studies, for both norethindrone and ethinylestradiol, the 90% CIs of AUC24 and Cmax on cycle day 6 before and after tazarotene administration were within the 80-125% boundary. In Study I, the 90% CIs of serum FSH and LH concentrations on cycle day 4 were within the 80-125% boundary. FSH and LH concentrations on cycle day 6 were marginally/partially outside the 80-125% boundary as a result of high variability. However, the mean FSH and LH serum concentrations on cycle day 6 of the third cycle were lower than those of the second cycle. In Study II, the 90% CIs of serum FSH, LH and progesterone concentrations were all within the 80-125% boundary, except for LH on cycle day 2. LH concentrations on cycle day 2 were marginally/partially outside the 80-125% boundary as a result of high variability. However, the mean serum LH concentration on cycle day 2 of the third cycle was lower than that of the second cycle. CONCLUSIONS: Oral tazarotene up to 6 mg once daily does not affect the pharmacokinetics and efficacy of Ortho-Novum 1/35.

Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry.

We investigated the formation of PGF(2alpha) 1-ethanolamide, PGE(2) 1-ethanolamide, and PGD(2) 1-ethanolamide (prostamides F(2alpha), E(2), and D(2), respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH -/-) male mice. One group of three normal mice was not dosed (naïve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH -/- mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F(2alpha), prostamide E(2), prostamide D(2), and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F(2alpha) was detected in tissues in FAAH -/- mice after administration of anandamide. Concentrations of anandamide, prostamide E(2), and prostamide D(2) in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH -/- mice than those of the anandamide-treated control mice. This report demonstrates that prostamides, including prostamide F(2alpha), were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking.

Evaluation of an immortalized retinal endothelial cell line as an in vitro model for drug transport studies across the blood-retinal barrier.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Intramuscular injection of 125I-botulinum neurotoxin-complex versus 125I-botulinum-free neurotoxin: time course of tissue distribution.

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.

Pharmacokinetics of tazarotene cream 0.1% after a single dose and after repeat topical applications at clinical or exaggerated application rates in patients with acne vulgaris or photodamaged skin.

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 microg/L. RESULTS: At exaggerated application rates in patients with acne vulgaris, the maximum average peak concentration (C(max)) and 24-hour area under the concentration-time curve (AUC) values of tazarotenic acid were (mean +/- SD) 1.20 +/- 0.41 microg/L (n = 10) and 17.0 +/- 6.1 microg. h/L (n = 10), respectively, and occurred on day 15. The single highest C(max) was 1.91 microg/L. At standard application rates in patients with acne vulgaris, the maximum average C(max) and AUC values of tazarotenic acid were 0.10 +/- 0.06 microg/L (n = 8) and 1.54 +/- 1.01 microg. h/L (n = 8), respectively, and occurred on day 15. At exaggerated application rates in patients with photodamaged skin, the maximum average C(max) and AUC values of tazarotenic acid were (mean +/- SD) 1.75 +/- 0.53 microg/L (n = 16) and 23.8 +/- 7.0 microg. h/L (n = 16), respectively, and occurred on day 22. The single highest C(max) was 3.43 microg/L on day 29. At standard application rates in patients with photodamaged skin, the maximum average C(max) and AUC values of tazarotenic acid were 0.236 +/- 0.255 microg/L (n = 8) and 2.44 +/- 1.38 microg. 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. CONCLUSIONS: 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 microg/L. The maximum average plasma concentrations of tazarotenic acid following application to an exaggerated body surface area (15%) were less than 1.8 microg/L.

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

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.

Blood concentrations of cyclosporin a during long-term treatment with cyclosporin a ophthalmic emulsions in patients with moderate to severe dry eye disease.

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.