Ocular Pharmacokinetics of Brimonidine Drug Delivery System in Monkeys and Translational Modeling for Selection of Dose and Frequency in Clinical Trials.

Brimonidine, a selective α 2-adrenoceptor agonist, displays putative retinal cyto- and neuroprotective activity in vitro and in vivo. An intravitreal sustained-release brimonidine implant, Brimonidine Posterior Segment Drug Delivery System (brimonidine DDS), allowing targeted drug delivery to the retina has been developed for potential clinical application. This study evaluates the in vivo posterior segment pharmacokinetics of brimonidine DDS implant in the monkey eye and applies translational pharmacokinetic modeling to predict tissue exposure in the human eye. Anesthetized cynomolgus monkeys received a single intravitreal injection of brimonidine DDS 400 µg implant before removal of study eyes at days 7, 30, 60, 92, 120, and 150 postimplant (three to four animals per time point) for assay of brimonidine in aqueous humor, vitreous, and retina samples. Brimonidine concentrations in the human eye were modeled using a linear, three-compartment model assuming bidirectional distribution to/from the aqueous humor and retina and elimination from the aqueous humor. Monkey tissue volumes were scaled up to human values; intercompartmental and elimination rate constants were assumed to be identical in the two species. Modeling and simulations were performed using NONMEM v. 7.3, R 3.5.1. Brimonidine exposure was highest in the monkey vitreous and retina; concentrations in the central (macula) and peripheral retina were maintained at high levels (>100 ng/g) for 3 to 4 months. Simulated brimonidine concentration-time profiles in human macula indicated that brimonidine DDS 400 µg implant would deliver effective drug concentrations (20.7‒82.2 ng/g, based on animal pharmacology) for approximately 3 months. Accordingly, administration of the 400 µg implant at 3-month intervals is recommended. SIGNIFICANCE STATEMENT: Brimonidine, an α 2-adrenoceptor agonist, is cyto- and neuroprotective in animal models of retinal/optic nerve injury. Brimonidine Posterior Segment Drug Delivery System (brimonidine DDS) is an intravitreal sustained-release implant with potential ophthalmological applications. This study explores the pharmacokinetics of brimonidine DDS 400 µg implant in the monkey eye and uses compartmental modeling to predict human ocular tissue exposure. Targeted retinal brimonidine delivery from vitreous was demonstrated in monkeys. Simulated tissue concentration-time profiles indicated persistence of pharmacologically effective brimonidine concentrations for ≈3 months in human retina.

A Mechanistic and Translational Pharmacokinetic-Pharmacodynamic Model of Abicipar Pegol and Vascular Endothelial Growth Factor Inhibition.

Abicipar pegol (abicipar) is a novel DARPin therapeutic and highly potent vascular endothelial growth factor (VEGF) inhibitor intended for the treatment of neovascular age-related macular degeneration (nAMD). Here we develop a translational pharmacokinetic/pharmacodynamic (PK/PD) model for abicipar to guide dosing regimens in the clinic. The model incorporated abicipar-VEGF binding kinetics, VEGF expression levels, and VEGF turnover rates to describe the ocular and systemic PK data collected from the vitreous, aqueous humor (AH), choroid, retina, and serum of rabbits after a 1-mg abicipar intravitreal (IVT) dose. The model was translated to humans using human-specific mechanistic parameters and refitted to human serum and AH concentrations from patients with diabetic macular edema and nAMD. The model was then used to simulate 8-, 12- (quarterly), and 16-week dosing intervals in the clinic. Simulations of 2 mg abicipar IVT at 8-week or quarterly dosing in humans indicates minimum steady-state vitreal concentrations are maintained above both in vitro IC50 and in vivo human IC50 values. The model predicted virtually complete VEGF inhibition for the 8-week and quarterly dosing schedule during the 52-week treatment period. In the 16-week schedule, clinically significant VEGF inhibition was maintained during the 52-week period. The model quantitatively described abicipar-VEGF target engagement leading to rapid reduction of VEGF and a long duration of VEGF inhibition demonstrating the clinical feasibility of up to a 16-week dosing interval. Abicipar is predicted to reduce IVT dosing compared with other anti-VEGF therapies with the potential to lessen patient treatment burden. SIGNIFICANCE STATEMENT: Current anti-VEGF treatments for neovascular age-related macular degeneration require frequent (monthly) intravitreal injections and monitoring, which increases patient burden. We developed a mechanistic pharmakinetic/pharmadynamic model to describe the interaction between abicipar (a novel VEGF inhibitor) and VEGF to evaluate the duration of action. The model demonstrates extended abicipar-VEGF target engagement leading to clinical feasibility of up to a 16-week dosing interval. Our model predicted that abicipar 8-week and quarterly dosing schedules maintain virtually complete VEGF inhibition during the 52-week period.

Clinical Pharmacokinetics of Oxymetazoline Cream Following Topical Facial Administration for the Treatment of Erythema Associated With Rosacea.

BACKGROUND: Oxymetazoline cream 1.0% is FDA-approved for the topical treatment of persistent facial erythema associated with rosacea in adults. This phase 2, multicenter, randomized, double-blind, parallel-group study assessed the pharmacokinetics, safety, and tolerability of oxymetazoline in patients with moderate to severe erythema associated with rosacea. METHODS: Eligible patients were randomized to 1 of 8 treatment groups (oxymetazoline cream 0.5%, 1.0%, or 1.5% or vehicle applied topically either once or twice daily for 28 days). Pharmacokinetic analyses were conducted in patients receiving oxymetazoline. Plasma samples for pharmacokinetic assessments were collected prior to dosing and 6 times postdose on days 1 and 28. RESULTS: A total of 356 patients were included in the safety population (oxymetazoline, n=268; vehicle, n=88). Thirty patients (11.2%) in the oxymetazoline group reported treatment-related treatment-emergent adverse events, most of which were mild to moderate application-site reactions. Oxymetazoline, at all concentrations, was generally safe and well tolerated. Mean maximum observed plasma concentrations were ≤115 pg/mL across all groups; the highest mean values for area under the plasma concentration-time curve from time 0 to 24 hours following once- and twice-daily administration of oxymetazoline 1.5% were 1680 pg•h/mL and 2660 pg•h/mL, respectively. Systemic exposure to oxymetazoline increased dose proportionally with once- and twice-daily administration. CONCLUSION: These findings support the use of oxymetazoline for the treatment of persistent facial erythema associated with rosacea. J Drugs Dermatol. 2018;17(2):213-220.

Assessment of tear film osmolarity using the TearLab™ osmometer in normal dogs and dogs with keratoconjunctivitis sicca.

OBJECTIVE: To evaluate repeatability and reproducibility of tear osmolarity measured using the TearLab™ osmometer in normal dogs and to assess its diagnostic potential in dogs with keratoconjunctivitis sicca (KCS). ANIMALS STUDIED: Beagle dogs; six normal and five with KCS. PROCEDURES: Tear osmolarity and Schirmer tear test-1 (STT-1) values were obtained at various times. Normal dogs were assessed for diurnal variation and repeatability and reproducibility of measurements. Dogs with KCS were evaluated before and after 5 months' topical twice-daily therapy with 2% cyclosporine. RESULTS: Mean ± SD tear osmolarity (mOsm/L) was significantly higher in normal dogs (337.4 ± 16.2) than in dogs with KCS before therapy (306.2 ± 18.0; P < 0.0001), but not following therapy with 2% cyclosporine (330.5 ± 13.7; P = 1.00). Osmolarity readings lower than 325.5 mOsm/L were suggestive of KCS (84.8% sensitivity and 87.1% specificity). In normal dogs, tear osmolarity readings were stable during the daytime (P = 0.99). Repeated measurements revealed high variability and typically poor-to-moderate repeatability and reproducibility, although this was improved by taking three successive measurements at each session. Considering combined data from all dogs, a positive correlation existed between STT-1 and tear osmolarity measurements (Pearson's correlation test, P = 0.04, r = 0.62). CONCLUSIONS: Canine tear osmolarity as determined by TearLab™ osmometer was variable, required multiple readings to be informative, and differed from values reported for humans. Dogs with KCS had a lower tear osmolarity than did normal dogs, and this increased following cyclosporine therapy.

Antibody-Drug Conjugate (ADC) Research in Ophthalmology--a Review.

Similar to cancer, many ocular proliferative disorders could be treated with a specific antibody conjugated to a toxin. Active targeting to inhibit epithelial and endothelial cell proliferation in the eye has been tested using antibody-drug conjugates (ADC) both pre-clinically and clinically. Achieving efficacious drug concentrations in the eye, in particular to treat back of the eye disorders is challenging, and the promise of targeted antibody mediated delivery holds great potential. In this review, we describe the research efforts in drug targeting using ADC for the treatment of choroidal neovascularization (CNV), posterior lens capsule opacification, and proliferative vitreoretinopathy. Among these disorders, CNV represents a more active research focus, with more target antigens tested, given the disease prevalence and wider target antigen selection based on current understanding of the pathophysiology of the disease. However, the only research advancing to testing in clinical stage is for posterior lens capsule opacification. Compared to oncology, ADC research and development in ophthalmology is much more limited, possibly due to availability of successful therapies that could be administered locally with limited concern of off-target drug toxicity.

The biodisposition and hypertrichotic effects of bimatoprost in mouse skin.

Studies on bimatoprost were performed with two objectives: (i) to determine whether bimatoprost possesses hair growth-stimulating properties beyond eyelash hypertrichosis and (ii) to investigate the biodisposition of bimatoprost in skin for the first time. Bimatoprost, at the dose used clinically for eyelash growth (0.03%) and given once daily for 14 days, increased pelage hair growth in C57/black 6 mice. This occurred as a much earlier onset of new hair growth in shaved mice and the time taken to achieve complete hair regrowth, according to photographic documentation and visual assessment. Bimatoprost biodisposition in the skin was determined at three concentrations: 0.01%, 0.03% and 0.06%. Dose-dependent C(max) values were obtained (3.41, 6.74, 12.3 µg/g tissue), and cutaneous bimatoprost was well maintained for 24 h following a single dose. Bimatoprost was recovered from the skin only as the intact molecule, with no detectable levels of metabolites. Thus, bimatoprost produces hypertrichosis as the intact molecule.

Neutralizing Antibody Formation with OnabotulinumtoxinA (BOTOX®) Treatment from Global Registration Studies across Multiple Indications: A Meta-Analysis.

Though the formation of neutralizing antibodies (NAbs) during treatment with botulinum neurotoxin is rare, their presence may nonetheless affect the biological activity of botulinum toxin and negatively impact clinical response. The goal of this updated meta-analysis was to evaluate and characterize the rate of NAb formation using an expanded dataset composed of 33 prospective placebo-controlled and open-label clinical trials with nearly 30,000 longitudinal subject records prior to and following onabotulinumtoxinA treatment in 10 therapeutic and aesthetic indications. Total onabotulinumtoxinA doses per treatment ranged from 10 U to 600 U administered in ≤15 treatment cycles. The NAb formation at baseline and post-treatment was tested and examined for impact on clinical safety and efficacy. Overall, 27 of the 5876 evaluable subjects (0.5%) developed NAbs after onabotulinumtoxinA treatment. At study exit, 16 of the 5876 subjects (0.3%) remained NAb positive. Due to the low incidence of NAb formation, no clear relationship was discernable between positive NAb results and gender, indication, dose level, dosing interval, treatment cycles, or the site of injection. Only five subjects who developed NAbs post-treatment were considered secondary nonresponders. Subjects who developed NAbs revealed no other evidence of immunological reactions or clinical disorders. This comprehensive meta-analysis confirms the low NAb formation rate following onabotulinumtoxinA treatment across multiple indications, and its limited clinical impact on treatment safety and efficacy.

Impact of Modifying Abicipar Manufacturing Process in Patients with Neovascular Age-Related Macular Degeneration: MAPLE Study Results.

Purpose: To evaluate the impact of modifying the abicipar pegol (abicipar) manufacturing process on the safety and treatment effect of abicipar in patients with neovascular age-related macular degeneration (nAMD). Methods: A new process for manufacturing abicipar was developed to reduce host cell impurities. In a prospective, Phase 2, multicenter, open-label, 28-week clinical trial, patients (n=123) with active nAMD received intravitreal injections of abicipar 2 mg at baseline (day 1) and weeks 4, 8, 16, and 24. Outcome measures included proportion of patients with stable vision (<15-letter loss from baseline; primary endpoint), change from baseline in best-corrected visual acuity (BCVA) and central retinal thickness (CRT), and adverse events. Results: Overall, 8.9% (11/123) of patients experienced intraocular inflammation (IOI) and discontinued treatment. IOI cases were assessed as mild (2.4% [3/123]), moderate (4.9% [6/123]), or severe (1.6% [2/123]) and resolved with steroid treatment. Visual acuity in most patients with IOI (8 of 11) recovered to baseline BCVA or better by study end. No cases of endophthalmitis or retinal vasculitis were reported. Stable vision was maintained for ≥95.9% (≥118/123) of patients at all study visits. At week 28, treatment-naïve patients showed a greater mean improvement from baseline in BCVA compared with previously treated patients (4.4 vs 1.8 letters) and a larger mean CRT reduction from baseline (98.5 vs 45.5 µm). Conclusion: Abicipar produced using a modified manufacturing process showed a moderately lower incidence and severity of IOI compared with Phase 3 abicipar studies. Beneficial effects of treatment were demonstrated.

Effect of Anti-VEGF Therapy on the Disease Progression of Neovascular Age-Related Macular Degeneration: A Systematic Review and Model-Based Meta-Analysis.

Anti-vascular endothelial growth factor (VEGF) therapy is used to slow the disease progression of neovascular age-related macular degeneration. Due to the treatment burden of frequent intravitreal injections, anti-VEGFs are often used on treat and extend protocols rather than the labeled frequency. The current goal of anti-VEGF drug development is to minimize treatment burden by reducing the number of intravitreal injections. The purpose of this systemic review and model-based meta-analysis (MBMA) was to (1) perform modeling to describe the disease progression of neovascular age-related macular degeneration in the absence of treatment, as well as in the presence of abicipar, aflibercept, brolucizumab, or ranibizumab intervention; (2) and to simulate virtual head-to-head comparisons among the drugs with an extended dose schedule of once every 12 weeks (Q12). Data sources were PubMed, internal Allergan data, www.clinicaltrials.gov, and www.clinicaltrialsregister.eu. Eligibility assessment was performed by 2 independent review authors. Randomized, controlled trials that had at least 1 arm with an anti-VEGF (aflibercept, abicipar, bevacizumab, brolucizumab, pegaptanib, or ranibizumab), a control arm of placebo or anti-VEGF, a treatment duration of at least 4 months, reported best-corrected visual acuity data, and at least 20 patients were included. A total of 22 trials, consisting of 55 arms, from across 9500+ subjects and 500+ best-corrected visual acuity observations were used to develop the model. Consistent with reported data, results from the model showed that abicipar Q12 underperformed ranibizumab (every 4 weeks), aflibercept (every 4 weeks), and brolucizumab (every 8 weeks/Q12) labeled dosing schedules. However, when all drugs were virtually tested using the extended schedule, abicipar outperformed ranibizumab and aflibercept and produced a similar week 52 change from baseline as brolucizumab. Predicted week 52 changes from baseline were 5.92 ± 1.02, 3.04 ± 1.61, 6.61 ± 0.284, and 3.02 ± 2.35 best-corrected visual acuity letters for abicipar, aflibercept, brolucizumab, and ranibizumab, respectively, using the Q12 schedule. Results demonstrate the feasibility of Q12 dosing with clinically meaningful letter gains for abicipar and brolucizumab. The model developed under this MBMA has utility for exploring different regimens for existing or novel anti-VEGF agents.

Nonclinical Pharmacokinetic and Pharmacodynamic Assessment of Bimatoprost Following a Single Intracameral Injection of Sustained-Release Implants.

Purpose: To assess the pharmacokinetic (PK)/pharmacodynamic (PD) relationship following intracameral Bimatoprost sustained-release (SR) implants (8, 15, 30, and 60 µg) in dogs to determine the optimal investigative dose in humans. Methods: Forty-four male normotensive beagle dogs were assigned to 1 of 8 groups receiving 8-, 15-, 30-, and 60-µg implants (PD assessment [n = 8/group, 4 groups]; PK assessment [n = 3/group, 4 groups]). Intraocular pressure (IOP) in PD animals and aqueous humor/blood concentrations of bimatoprost and its acid in PK animals were assessed. PK/PD correlation analysis was performed using steady-state data. Residual implants were recovered to assess polymer degradation. Results: Dose-dependent IOP lowering was observed for all dose groups for at least 3 months postdose. Blood concentrations of bimatoprost and bimatoprost acid were below the limit of quantification (<0.25 ng/mL), whereas dose-dependent concentration-time profiles were observed in the aqueous humor. At steady state, observed and predicted correlation between aqueous humor drug concentration and IOP lowering was similar and translatable to findings in humans following topical bimatoprost eyedrop administration. Implants at all doses were well tolerated and polymer degradation was apparent. Conclusions: Dose-dependent IOP lowering with Bimatoprost SR was maintained for at least 3 months in dogs, and the implants were well tolerated. The established PK/PD relationship appears to translate to humans. Doses between 8 and 15 µg appear to provide the best benefit/risk profile for clinical development of the implants. Translational Relevance: The close PK/PD relationship between dog and human helps inform which bimatoprost dose should be investigated in clinical studies.

Distribution of 14C-Latanoprost Following a Single Intracameral Administration Versus Repeated Topical Administration.

PURPOSE: To qualitatively evaluate the ocular and periocular distribution of 14C-latanoprost following a single intracameral administration or repeated topical ocular administration in beagle dogs and cynomolgus monkeys. METHODS: In the dog study, three animals received an intracameral dose of 14C-latanoprost bilaterally and were euthanized at 1, 2, and 4 h post dose; three control animals received topical 14C-latanoprost bilaterally once daily for 5 days and were euthanized at 1, 4, and 24 h post final dose. Sagittal 40-µm sections of eyes with surrounding tissues were collected and processed for autoradiography. Methods in the monkey study were similar; two animals received a unilateral intracameral dose of 14C-latanoprost. RESULTS: After intracameral dosing in dogs, radioactivity was concentrated in the cornea, iris, ciliary body, and anterior chamber with no radioactivity detected in the eyelids or other periorbital tissues. After topical dosing, radioactivity was distributed in the bulbar conjunctiva, cornea, anterior chamber, iris, ciliary body, upper and lower eyelids, and periorbital tissues (fat/muscle). After intracameral dosing in monkeys, radioactivity was concentrated in the anterior chamber, cornea, iris, ciliary body, and posteriorly along the uveoscleral outflow pathway; there was no radioactivity in the eyelids or periorbital tissues aside from signal in the nasolacrimal duct, likely from reflux of 14C-latanoprost into the tear film. CONCLUSIONS: Intracameral delivery resulted in more selective target tissue drug exposure. Intracameral drug delivery has potential to reduce ocular surface and periocular adverse effects associated with topical administration of prostaglandin analogues, such as eyelash growth and periorbital fat atrophy.

Intracameral Sustained-Release Bimatoprost Implant Delivers Bimatoprost to Target Tissues with Reduced Drug Exposure to Off-Target Tissues.

PURPOSE: To explore the ocular distribution of bimatoprost after intracameral administration of a biodegradable sustained-release bimatoprost implant (Bimatoprost SR) versus repeated topical administration of bimatoprost 0.03% ophthalmic solution in dogs. Bimatoprost SR and topical bimatoprost 0.03% previously were shown to have similar intraocular pressure-lowering effects in humans in a phase 1/2 clinical trial. METHODS: Twenty-four beagle dogs received either once-daily topical bimatoprost 0.03% for 7 days or a bilateral intracameral administration of Bimatoprost SR (15 µg). At predetermined time points, ocular tissues were collected and concentrations of bimatoprost and bimatoprost acid were quantified using liquid chromatography-tandem mass spectrometry. RESULTS: Bimatoprost SR administration enhanced delivery of study drug to a site of action [iris-ciliary body (ICB)] compared with topical bimatoprost (Cmax [bimatoprost+bimatoprost acid] = 18,200 and 4.13 ng/g, respectively). However, distribution of drug to tissues associated with prostaglandin analog (PGA)-related side effects (i.e., bulbar conjunctiva, eyelid margins, and periorbital fat) was limited following Bimatoprost SR administration (Cmax [bimatoprost+bimatoprost acid] = BLQ [beneath the limit of quantitation] to 0.354 ng/g) compared with topical dosing (Cmax [bimatoprost+bimatoprost acid] = 36.6-2,110 ng/g). CONCLUSIONS: Bimatoprost SR administration in dogs selectively delivered drug to the ICB with low or undetectable drug levels in ocular surface and extraocular tissues. Use of Bimatoprost SR for glaucoma treatment may reduce the incidence of adverse events typically associated with topical PGAs by targeting bimatoprost delivery to the key site of action of the PGA class and reducing exposure to off-target tissues.

Design and Conduct Considerations for First-in-Human Trials.

A milestone step in translational science to transform basic scientific discoveries into therapeutic applications is the advancement of a drug candidate from preclinical studies to initial human testing. First-in-human (FIH) trials serve as the link to advance new promising drug candidates and are conducted primarily to determine the safe dose range for further clinical development. Cross-functional collaboration is essential to ensure efficient and successful FIH trials. The aim of this publication is to serve as a tutorial for conducting FIH trials for both small molecule and biological drug candidates with topics covering regulatory requirements, preclinical safety testing, study design considerations, safety monitoring, biomarker assessment, and global considerations. An emphasis is placed on FIH trial design considerations, including starting dose selection, study size and population, dose escalation scheme, and implementation of adaptive designs. In light of the recent revision of the European Medicines Agency (EMA) guideline on FIH trials to promote safety and mitigate risk, we also discuss new measures introduced in the guideline that impact FIH trial design.

Ocular pharmacokinetics and tolerability of bimatoprost ophthalmic solutions administered once or twice daily in rabbits, and clinical dosing implications.

PURPOSE: Fixed-combination medications can benefit patients requiring multiple agents to lower their intraocular pressure (IOP), but combining agents with complementary mechanisms of action is challenging if their dosing frequency differs. This study compares in vivo pharmacokinetic and ocular tolerability of bimatoprost 0.01% ophthalmic solutions dosed once or twice daily. Reports of twice-daily dosing in glaucoma patients are also reviewed. METHODS: New Zealand White rabbits were administered bimatoprost 0.01% monotherapy or fixed-combination bimatoprost 0.01%/brimonidine 0.1%, once or twice daily in both eyes for 4 days. Ocular tissues were harvested and analyzed by liquid chromatography-tandem mass spectrometry. The pharmacokinetic parameters calculated included maximum observed concentration, time to maximum concentration, and area under the concentration-time curve. RESULTS: Due to extensive metabolism, bimatoprost concentration was below the quantitation limit by 1 hour post-dose in all samples. Bimatoprost acid exposure, however, could be measured up to 6-8 hours post-dose and was similar in the aqueous humor and iris-ciliary body (pharmacological site of action) of animals treated once or twice daily with either bimatoprost 0.01% or fixed-combination bimatoprost 0.01%/brimonidine 0.1%. Increasing dosage frequency in rabbits did not raise the incidence of drug-related conjunctival hyperemia (most common adverse event associated with bimatoprost use in humans), suggesting comparable ocular tolerability of the once- and twice-daily regimens for each formulation. CONCLUSION: Bimatoprost 0.01% administered once or twice daily as monotherapy and in fixed-combination with brimonidine 0.1% in rabbits show similar pharmacokinetic profiles of bimatoprost acid, especially in the iris-ciliary body. Key findings from previous clinical studies suggest that by varying the concentration of benzalkonium chloride (a preservative with corneal penetration-enhancing properties), formulations of bimatoprost 0.01% can be administered once or twice daily. These findings support development of bimatoprost 0.01%-based fixed-dose combination therapies administered twice daily for patients who require multiple adjunctive medications to control their IOP.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes.

PURPOSE: To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. METHODS: Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1°ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). RESULTS: Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1°ATD-eyes and exacerbated 1°ATD-LG atrophy. IMIP were evident in 2°ATD-LG as well as 1°ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. CONCLUSIONS: The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms.

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.

Ophthalmic drug delivery considerations at the cellular level: drug-metabolising enzymes and transporters.

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.

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.