Topical pharmacokinetics of brinzolamide suspensions in rabbits and variability analysis for sample size and design considerations.

Identifying critical attributes for complex locally acting ophthalmic formulations and establishing in vitro-in vivo correlations can facilitate selection of appropriate thresholds for formulation changes that reflect lack of impact on in vivo performance. In this study the marketed antiglaucoma product Azopt® (1% brinzolamide suspension) and five other brinzolamide formulations varying in particle size distributions and apparent viscosities were topically administered in rabbits, and their ocular pharmacokinetics was determined in multiple ocular tissues. Statistical evaluation with ANOVA showed no significant differences between the formulations in the peak drug concentration (Cmax) in the aqueous humor and iris-ciliary body. As a post-hoc analysis, the within animal and total variability was determined for Cmax in the aqueous humor and iris-ciliary body. Based on the observed variability, we investigated the sample size needed for two types of study designs to observe statistically significant differences in Cmax. For the sample size calculations, assuming both 25% and 50% true differences in Cmax between two formulations, two study designs were compared: paired-eye dosing design (one formulation in one eye and another formulation in the other eye of the same animal at the same time) versus parallel-group design. The number of rabbits needed in the paired-eye dosing design are much lower than in the parallel-group design. For example, when the true difference in aqueous humor Cmax is 25%, nine rabbits are required in the paired-eye design versus seventy rabbits (35 per treatment) in the parallel-group design to observe a statistically significant difference with a power of 80%. Therefore, the proposed paired-eye dosing design is a viable option for the design of pharmacokinetic studies comparing ophthalmic products to determine the impact of formulation differences.

Characterization and validation of a chronic retinal neovascularization rabbit model by evaluating the efficacy of anti-angiogenic and anti-inflammatory drugs.

AIM: To establish a rabbit model with chronic condition of retinal neovascularization (RNV) induced by intravitreal (IVT) injection of DL-2-aminoadipic acid (DL-AAA), a retinal glial (Müller) cell toxin, extensive characterization of DL-AAA induced angiographic features and the suitability of the model to evaluate anti-angiogenic and anti-inflammatory therapies for ocular vascular diseases. METHODS: DL-AAA (80 mmol/L) was administered IVT into both eyes of Dutch Belted rabbit. Post DL-AAA delivery, clinical ophthalmic examinations were performed weekly following modified McDonald-Shadduck Scoring System. Color fundus photography, fluorescein angiography (FA), and optical coherence tomography (OCT) procedures were performed every 2 or 4wk until stable retinal vascular leakage was observed. Once stable retinal leakage (12wk post DL-AAA administration) was established, anti-vascular endothelial growth factor (VEGF) (bevacizumab, ranibizumab and aflibercept) and anti-inflammatory (triamcinolone, TAA) drugs were tested for their efficacy after IVT administration. Fluorescein angiograms were scored before and after treatment following a novel grading system, developed for the DL-AAA rabbit model. RESULTS: Post DL-AAA administration, eyes were presented with moderate to severe retinal/choroidal inflammation which was accompanied by intense vitreous flare and presence of inflammatory cells in the vitreous humor. Retinal hemorrhage was restricted to the tips of neo-retinal vessels. FA revealed maximum retinal vascular leakage at 2wk after DL-AAA injection and then persisted as evidenced by stable mean FA scores in weeks 8 and 12. Retinal vascular angiographic and tomographic features were stable and consistent up to 36mo among two different staggers induced for RNV at two different occasions. Day 7, mean FA scores showed that 1 µg/eye of bevacizumab, ranibizumab, aflibercept and 2 µg/eye of TAA suppress 65%, 90%, 100% and 50% retinal vascular leakage, respectively. Day 30, bevacizumab and TAA continued to show 66% and 44% suppression while ranibizumab effect was becoming less effective (68%). In contrast, aflibercept was still able to fully (100%) suppress vascular leakage on day 30. On day 60, bevacizumab, ranibizumab and TAA showed suppression of 7%, 12%, and 9% retinal vascular leakage, respectively, however, aflibercept continued to be more effective showing 50% suppression of vascular leakage. CONCLUSION: The DL-AAA rabbit model mimics RNV angiographic features like RNV and chronic retinal leakage. Based on these features the DL-AAA rabbit model provides an invaluable tool that could be used to test the therapeutic efficacy and duration of action of novel anti-angiogenic formulations, alone or in combination with anti-inflammatory compounds.

Topical pharmacokinetics of dexamethasone suspensions in the rabbit eye: Bioavailability comparison.

Topical corticosteroids are used to treat inflammation of the anterior segment. Due to their low water-solubility, they are often formulated as suspensions, but ocular bioavailability of the suspensions is not known. Herein, ocular pharmacokinetics of dexamethasone in albino rabbits was investigated following intracameral administration of dexamethasone solution and topical administration of three commercial suspensions: Maxidex®, TobraDex®, and TobraDexST®. Dexamethasone concentrations in tear fluid, cornea, aqueous humor, conjunctiva and iris-ciliary body were determined. Non-compartmental analysis was performed to estimate the pharmacokinetic parameters of dexamethasone. Following intracameral administration, the clearance and the apparent volume of distribution were estimated to be 13.6 µL/min and 990 µL, respectively. After topical administration, the absolute aqueous humor bioavailability for dexamethasone (<2%) is being reported for the first time. The highest value was obtained for TobraDexST® followed by Maxidex® and TobraDex®. This study provides for the first-time comprehensive and quantitative ocular pharmacokinetic parameters (including absolute bioavailability) for topically instilled dexamethasone suspensions. Furthermore, the new intracameral pharmacokinetic parameters allow a rational and quantitative basis for the design of improved ocular dexamethasone delivery systems.

Principles and Experimental Considerations for In Vitro Transporter Interaction Assays.

Drug transporters are universally acknowledged as important determinants of the absorption, distribution, metabolism, and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design, will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.

Case Study 7: Transporters Case Studies-In Vitro Solutions for Translatable Outcomes.

Assessing the interactions of a new drug candidate with transporters, either as a substrate, inhibitor, or inducer, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to 11 for the EMA as of 2020. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the most relevant tools for the study and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter are unpublished at the time of the compilation of this book. It has been included in this chapter to provide a sense of the complexities in transporter kinetics to the reader.

Comprehensive Ocular and Systemic Pharmacokinetics of Brinzolamide in Rabbits After Intracameral, Topical, and Intravenous Administration.

Brinzolamide is a topical carbonic anhydrase inhibitor which reduces the production of aqueous humor in the ciliary body, thereby reducing intra-ocular pressure. It is formulated as an ophthalmic suspension. The pharmacokinetics of ocular suspensions is not well understood. The objective of this study was to characterize the pharmacokinetics of brinzolamide in rabbit aqueous humor, iris-ciliary body, plasma, and whole blood. New Zealand White rabbits were dosed via intracameral, topical and intravenous administration. After intracameral administration (4.5 µg) of solubilized brinzolamide, aqueous humor concentrations were described with a two-compartment model, the estimated clearance was 4.12 µL/min, apparent volume of distribution at steady-state 673 µL, and terminal half-life 3.4 h. After topical administration of 1% brinzolamide suspension (500 µg), absolute bioavailability based on aqueous humor AUC0-∞ was 0.10%. After intravenous administration of brinzolamide solution (0.75 mg/kg) elimination half-life in plasma and whole blood appeared to be over two weeks. The ratios of the measured concentrations of irisciliary body to whole blood, to plasma, and to aqueous humor concentrations enabled direct comparisons, and helped identify the significant contribution of the conjunctival-scleral pathways of absorption to the ciliary body. This study shows for the first-time the absolute bioavailability in aqueous humor and provides comprehensive pharmacokinetic parameters following administration of a topical suspension.

Pharmacokinetics and Pharmacodynamics of Immediate- and Modified-Release Mycophenolic Acid Preparations in Healthy Beagle Dogs.

Background: Mycophenolic acid (MPA) is a broad-acting immunomodulating agent that may be therapeutically beneficial for the treatment of immune-mediated diseases in canine patients. Objectives: To determine the suppressive effects of MPA on T-cell proliferation, and to assess the feasibility of a canine-specific q24 h modified-release MPA formulation (OKV-1001b). Animals: Fifteen healthy purpose-bred male beagle dogs. Methods: Two nearly identical open-label fifteen-day studies were conducted in which dogs were randomized to receive mycophenolate mofetil (MMF; 10 mg/kg q12h), or two doses of OKV-1001b (270 mg and 180 mg; q24h). Serial pharmacokinetic (PK) and pharmacodynamic (PD) samples were collected on Days 1, 8, and 15. MPA plasma concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), while an ex vivo T-cell proliferation assay assessed PD effects. Dogs were continuously monitored for evidence of side effects and gastrointestinal tolerability. Results: MPA induced inhibition of T-cell proliferation was observed following administration of all MPA preparations in a clear concentration-dependent manner. The PK/PD relationship was maintained across all days and time-points. Data generated herein suggest that MPA plasma concentrations above 600 ng/mL achieve at least 50% inhibition of T-cell proliferation. Conclusions and Clinical Importance: MPA holds therapeutic potential for treating dogs with immune-mediated disease, but clinical trials will be necessary to determine its safety and efficacy in naturally occurring disease. Likewise, q24h oral modified release MPA preparations that maintain MPA plasma concentrations between 600 and 1,000 ng/mL are warranted for further studies in client-owned dogs.

Principles and experimental considerations for in vitro transporter interaction assays.

Drug transporters are now universally acknowledged as important determinants of the absorption, distribution, metabolism and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.

Case study 6. Transporter case studies: in vitro solutions for translatable outcomes.

Assessing the interactions of a new drug candidate with transporters, either as a substrate or as an inhibitor, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to eleven for the EMA as of 2013. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to really appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the best tools for the job and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in transporter kinetics to the reader.