Qualification of In Vitro Dissolution Absorption System 2 (IDAS2) with Caco-2 and MDCK Cell Monolayers: Dose Sensitivity Study Using BCS Class I and III Drugs.

This study aimed to validate the In vitro Dissolution Absorption System 2 (IDAS2) containing a biological barrier of Caco-2 or Madin-Darby canine kidney (MDCK) cell monolayer through dose sensitivity studies. Metoprolol and propranolol were selected as Biopharmaceutics Classification System (BCS) Class I model drugs, and atenolol as a Class III model drug. The IDAS2 is comprised of a dissolution vessel (500 mL) and two permeation chambers (2 × 8.0 mL) mounted with Caco-2 or MDCK cell monolayer. One or two immediate-release tablet(s) of the model drug were added to the dissolution vessel, and the time profiles of dissolution and permeation were observed. Greater than 85% of metoprolol and propranolol (tested at two dosing concentrations) were dissolved by 15 min, and all drugs were fully dissolved by 30 min. All three drugs were more permeable across Caco-2 cells than MDCK cells with a linear increase in permeation across both cells at both dose concentrations. Thus, the dose sensitivity of the IDAS2 was demonstrated using both cell barriers. These results indicate a successful qualification of IDAS2 for the development/optimization of oral formulations and that MDCK cells can be utilized as a surrogate for Caco-2 cells.

The impact of surface area per volume (SA/V) ratio on drug transport from supersaturated solutions of ketoconazole.

The purpose of this study aimed to evaluate the impact of the surface area per volume (SA/V) ratio on drug transport from two supersaturated solutions (SSs) of ketoconazole with and without hydroxypropyl methylcellulose (HPMC), used as a precipitation inhibitor. In vitro dissolution, membrane permeation with two SA/V ratios, and in vivo absorption profiles for both SSs were determined. For the SS without HPMC, a two-step precipitation process due to the liquid-liquid phase separation was observed; the constant concentration with approximately 80 % of the dissolved amount was maintained for the first 5 min and subsequently decreased between 5 and 30 min. For the SS with HPMC, a parachute effect was observed; the constant concentration with approximately 80 % dissolved amount was maintained for more than 30 min and decreased very slowly thereafter. Assessment of the SA/V ratio using in vitro and in vivo models demonstrated that when the SA/V ratio was small, the SS with HPMC resulted in a significantly higher permeated amount than the SS without HPMC. In contrast, when the SA/V ratio was large, the HPMC-mediated parachute effect on drug transport from SSs was attenuated, both in vitro and in vivo. The parachute effect by HPMC decreased as the SA/V ratio increased, and the performance of supersaturating formulations would be overestimated by in vitro studies with small SA/V ratios.

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.

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.

Compartmental models for apical efflux by P-glycoprotein--part 1: evaluation of model complexity.

PURPOSE: With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data. METHODS: Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated. Several models were evaluated, and characteristics including membrane configuration, lipid content, and apical surface area (asa) were varied. RESULTS: Passive permeabilities from MDCK-MDR1 cells in the presence of cyclosporine gave lower model errors than from MDCK control cells. Consistent with the results in Part 2, model configuration had little impact on calculated model errors. The 5-compartment model was the simplest model that reproduced experimental lag times. Lipid content and asa had minimal effect on model errors, predicted lag times, and intracellular concentrations. Including endogenous basolateral uptake activity can decrease model errors. Models with and without explicit membrane barriers differed markedly in their predicted intracellular concentrations for basolateral drug exposure. Single point data resulted in clearances similar to time course data. CONCLUSIONS: Compartmental models are useful to evaluate the impact of efflux transporters on intracellular concentrations. Whereas a 3-compartment model may be sufficient to predict the impact of transporters that efflux drugs from the cell, a 5-compartment model with explicit membranes may be required to predict intracellular concentrations when efflux occurs from the membrane. More complex models including additional compartments may be unnecessary.

Models to predict unbound intracellular drug concentrations in the presence of transporters.

Knowledge of free drug intracellular concentration is necessary to predict the impacts of drugs on intracellular targets. The goal of this study was to develop models to predict free intracellular drug concentrations in the presence of apical efflux transporters. The apical efflux transporter P-glycoprotein (P-gp), encoded by human gene multidrug resistance 1 (MDR1), was studied. Apparent permeabilities for 10 compounds in Madin-Darby canine kidney (MDCK) and MDR1-MDCK cell monolayers were obtained experimentally. Six of these compounds were evaluated additionally in the presence of the P-gp inhibitor cyclosporine A. A three-compartment model was developed, and passive and apical efflux clearances (CL(d) and CL(ae), respectively) were estimated. Endogenous canine transporters also were delineated. The three-compartment model was unable to simulate experimentally observed lag times and exhibited systematic bias across the simulations. Next, a five-compartment model with explicit membrane compartments was developed. This model resulted in lower systematic errors and simulated the lag time observed experimentally. Apical efflux was modeled out of the cell or out of the membrane. The five-compartment model with apical efflux out of the membrane predicted marked differences in unbound intracellular concentrations between the apical-to-basolateral and the basolateral-to-apical directions. Upon apical drug addition, large decreases in intracellular concentrations were observed with the efflux transporter. No such difference was predicted upon basolateral drug addition. This is consistent with experimental differences in the impact of P-gp on hepatic and brain distribution and supports the hypothesis that apical efflux occurs out of the apical membrane.

Effects of human immunodeficiency virus protease inhibitors on the intestinal absorption of tenofovir disoproxil fumarate in vitro.

Human immunodeficiency virus protease inhibitors (PIs) modestly affect the plasma pharmacokinetics of tenofovir (TFV; -15% to +37% change in exposure) following coadministration with the oral prodrug TFV disoproxil fumarate (TDF) by a previously undefined mechanism. TDF permeation was found to be reduced by the combined action of ester cleavage and efflux transport in vitro. Saturable TDF efflux observed in Caco-2 cells suggests that at pharmacologically relevant intestinal concentrations, transport has only a limited effect on TDF absorption, thus minimizing the magnitude of potential intestinal drug interactions. Most tested PIs increased apical-to-basolateral TDF permeation and decreased secretory transport in MDCKII cells overexpressing P-glycoprotein (Pgp; MDCKII-MDR1 cells) and Caco-2 cells. PIs were found to cause a multifactorial effect on the barriers to TDF absorption. All PIs showed similar levels of inhibition of esterase-dependent degradation of TDF in an intestinal subcellular fraction, except for amprenavir, which was found to be a weaker inhibitor. All PIs caused a dose-dependent increase in the accumulation of a model Pgp substrate in MDCKII-MDR1 cells. Pgp inhibition constants ranged from 10.3 microM (lopinavir) to >100 microM (amprenavir, indinavir, and darunavir). Analogous to hepatic cytochrome P450-mediated drug interactions, we propose that the relative differences in perturbations in TFV plasma levels when TDF is coadministered with PIs are based in part on the net effect of inhibition and induction of intestinal Pgp by PIs. Combined with prior studies, these findings indicate that intestinal absorption is the mechanism for changes in TFV plasma levels when TDF is coadministered with PIs.

Evaluation of volatile ion-pair reagents for the liquid chromatography-mass spectrometry analysis of polar compounds and its application to the determination of methadone in human plasma.

A liquid chromatography method using volatile ion-pairing reagents and tandem mass spectrometry was developed to obviate observed matrix effect for ionizable polar compounds. The present study investigated the addition of volatile ion-pair reagents to the reconstitution solution instead of the mobile phase to enhance the efficiency of chromatographic separation and minimize the sensitivity loss due to the formation of ion-pairs. The volatile ion-pair reagents used were perfluorinated carboxylic acids with n-alkyl chains: heptafluorobutanoic acid (HFBA), nonafluoropentanoic acid (NFPA), tridecafluoroheptanoic acid (TDFHA) and pentadecafluorooctanoic acid (PDFOA). The model analytes evaluated were N-methylnicotinamide (MNA) chloride, N-methyl 2-pyridone 5-carboxamide (2PY) and phenylephrine. The effects of alkyl chain length and the concentrations of the ion-pair reagents on the retention of analytes were studied, as well as the effect of pH on the retention of phenylephrine. The volatile ion-pair reagents in the reconstitution solution showed significant effect on the retention of the ionizable polar compounds, and the sensitivity of detection was improved for plasma samples through decreasing the matrix effect. This methodology was successfully applied to establish a quantitative assay for the polar drug substance methadone in human plasma with a concentration range from 0.1 to 50 ng/mL. Ion-pair reagents not only shifted the retention time but also reduced the carry-over peak for methadone.