Utilization of In Vitro, In Vivo and In Silico Tools to Evaluate the pH-Dependent Absorption of a BCS Class II Compound and Identify a pH-Effect Mitigating Strategy.

PURPOSE: To describe a stepwise approach to evaluate the pH effect for a weakly basic drug by in vitro, in vivo and in silico techniques and identify a viable mitigation strategy that addresses the risk. METHODS: Clinical studies included assessment of the pH effect with famotidine. In vitro dissolution was evaluated in various biorelevant media and in a pH-shift test. PK studies in dogs were conducted under pentagastrin or famotidine pre-treatment and GastroPlus was employed to model human and dog PK data and simulate the performance in human. RESULTS: Clinical data indicated considerable pH dependent absorption of the drug when dosed in the presence of H2-antagonists. In vitro dissolution and in vivo dog data confirmed that the observed pH effect was due to reduced dissolution rate and lower solubility at increased gastric and intestinal pH. A salt form was identified to overcome the effect by providing fast dissolution and prolonged supersaturation. GastroPlus simulations predicted a mitigation of the pH effect by the salt. CONCLUSIONS: The drug exhibited a strong pH-effect in humans. The in vitro, in vivo and modeling approach provides a systematic workflow to evaluate the risk of a new drug and identify a strategy able to mitigate the risk.

Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations.

Protein therapeutics represent a rapidly growing proportion of new medicines being developed by the pharmaceutical industry. As with any new drug, an Occupational Exposure Limit (OEL) should be developed to ensure worker safety. Part of the OEL determination addresses bioavailability (BA) after inhalation, which is poorly understood for protein therapeutics. To explore this, male Sprague-Dawley rats were exposed intravenously or by nose-only inhalation to one of five test proteins of varying molecular size (10-150 kDa), including a polyethylene glycol-conjugated protein. Blood, lung tissue and bronchoalveolar lavage (BAL) fluid were collected over various time-points depending on the expected test protein clearance (8 minutes-56 days), and analyzed to determine the pharmacokinetic profiles. Since the BAL half-life of the test proteins was observed to be > 4.5 h after an inhalation exposure, accumulation and direct lung effects should be considered in the hazard assessment for protein therapeutics with lung-specific targets. The key finding was the low systemic bioavailability after inhalation exposure for all test proteins (∼≤1%) which did not appear molecular weight-dependent. Given that this study examined the inhalation of typical protein therapeutics in a manner mimicking worker exposure, a default 1% BA assumption is reasonable to utilize when calculating OELs for protein therapeutics.

Characterization of a Liquid Crystal System for Sustained Release of a Peptide BMS-686117.

Liquid crystal lipid-based formulations are an effective approach to prolong pharmacokinetics and reduce burst release of a drug on subcutaneous delivery. The objective of this paper was to investigate the influence of phase structures of a lipid-based liquid crystal delivery system and its associated mechanical properties on the release profile of a peptide. It was hypothesized that release of drug molecules are closely related to the mechanical properties that are controlled by phase structures. Experimentally, the relationship between phase structures of lipid liquid crystal system-soy phosphatidyl choline (SPC) and glycerol dioleate (GDO) in water were characterized by polarized light microscopy and small angle X-ray diffraction. Their rheological properties were evaluated with a rheometer and the in vitro release of the peptide as a measure drug release from the LC-depot injection. Three phases: disordered phase, lamellar phase, mixtures of cubic, lamellar, and hexagonal phases were detected by varying formulation compositions. A significant difference in rheological behavior was observed. The disordered phase displayed some attributes of typical Newtonian fluid with lowest viscosity while the lamellar phase showed a shear thinning behavior. Regarding the mechanical strength, the lamellar phase presents the highest storage modulus due to its layer structure followed by mixed phases. Comparing release profiles, the lamellar phase produced a fast release followed by the mixture of phases. In conclusion, this study demonstrates the ability to characterize LC phase structures with microscopy, small angle X-ray diffraction, and rheological measurements and their link to modulating a peptide release profile.

Mitigation of Adverse Clinical Events of a Narrow Target Therapeutic Index Compound through Modified Release Formulation Design: An in Vitro, in Vivo, in Silico, and Clinical Pharmacokinetic Analysis.

BMS-914392 is a tricyclic pyranoquinoline BCS class 2 weak base that demonstrates high solubility in low pH environments. Initial clinical studies indicated that rapid release of high dose BMS-914392 led to transient adverse events associated with peak plasma concentrations. A modified release (MR) formulation strategy was proposed to suppress the peak blood concentration and maintain total exposure to overcome the adverse effects. Three modified release prototype formulations were developed and tested via a USP 3 dissolution method to verify that each formulation can effectively slow the release of BMS-914392. A pharmacokinetic (PK) absorption model was employed to guide the formulation development and selection. Simulations showed good agreement with plasma levels measured after oral dosing in dogs. Identification of key formulation factors to achieve release rates suitable for blunting peak blood levels without diminishing exposure were achieved through combined preclinical data and use of GastroPlus simulations. PK absorption model refinements based on phase 1 data, dog pharmacokinetic results, and in vitro data provided reliable predictions of human absorption profiles and variability in patients. All three prototype formulations demonstrated lower maximum plasma concentrations of BMS-914392 and maintained satisfactory relative bioavailability. Both the PK absorption model and subsequent clinical data indicated that an acidified hydrophilic matrix MR formulation had the greatest potential to reduce the incidence of adverse events and showed the best exposure profile in fasted state healthy subjects with and without famotidine coadministration. The risk based development process achieved successful screening and selection of a suitable modified release formulation to enable clinical efficacy trials.

From bench to humans: formulation development of a poorly water soluble drug to mitigate food effect.

This study presents a formulation approach that was shown to mitigate the dramatic food effect observed for a BCS Class II drug. In vitro (dissolution), in vivo (dog), and in silico (GastroPlus®) models were developed to understand the food effect and design strategies to mitigate it. The results showed that such models can be used successfully to mimic the clinically observed food effect. GastroPlus® modeling showed that food effect was primarily due to the extensive solubilization of the drug into the dietary lipid content of the meal. Several formulations were screened for dissolution rate using the biorelevant dissolution tests. Surfactant type and binder amount were found to play a significant role in the dissolution rate of the tablet prototypes that were manufactured using a high-shear wet granulation process. The performance of the lead prototypes (exhibiting best in vitro dissolution performance) was tested in dogs and human subjects. A new formulation approach, where vitamin E TPGS was included in the tablet formulation, was found to mitigate the food effect in humans.

Towards more tolerable subcutaneous administration: Review of contributing factors for improving combination product design.

Subcutaneous (SC) injections can be associated with local pain and discomfort that is subjective and may affect treatment adherence and overall patient experience. With innovations increasingly focused on finding ways to deliver higher doses and volumes (≥2 mL), there is a need to better understand the multiple intertwined factors that influence pain upon SC injection. As a priority for the SC Drug Development & Delivery Consortium, this manuscript provides a comprehensive review of known attributes from published literature that contribute to pain/discomfort upon SC injection from three perspectives: (1) device and delivery factors that cause physical pain, (2) formulation factors that trigger pain responses, and (3) human factors impacting pain perception. Leveraging the Consortium's collective expertise, we provide an assessment of the comparative and interdependent factors likely to impact SC injection pain. In addition, we offer expert insights and future perspectives to fill identified gaps in knowledge to help advance the development of patient-centric and well tolerated high-dose/high-volume SC drug delivery solutions.

Assessing the risk of pH-dependent absorption for new molecular entities: a novel in vitro dissolution test, physicochemical analysis, and risk assessment strategy.

Weak base therapeutic agents can show reduced absorption or large pharmacokinetic variability when coadministered with pH-modifying agents, or in achlorhydria disease states, due to reduced dissolution rate and/or solubility at high gastric pH. This is often referred to as pH-effect. The goal of this study was to understand why some drugs exhibit a stronger pH-effect than others. To study this, an API-sparing, two-stage, in vitro microdissolution test was developed to generate drug dissolution, supersaturation, and precipitation kinetic data under conditions that mimic the dynamic pH changes in the gastrointestinal tract. In vitro dissolution was assessed for a chemically diverse set of compounds under high pH and low pH, analogous to elevated and normal gastric pH conditions observed in pH-modifier cotreated and untreated subjects, respectively. Represented as a ratio between the conditions, the in vitro pH-effect correlated linearly with clinical pH-effect based on the Cmax ratio and in a non-linear relationship based on AUC ratio. Additionally, several in silico approaches that use the in vitro dissolution data were found to be reasonably predictive of the clinical pH-effect. To explore the hypothesis that physicochemical properties are predictors of clinical pH-effect, statistical correlation analyses were conducted using linear sequential feature selection and partial least-squares regression. Physicochemical parameters did not show statistically significant linear correlations to clinical pH-effect for this data set, which highlights the complexity and poorly understood nature of the interplay between parameters. Finally, a strategy is proposed for implementation early in clinical development, to systematically assess the risk of clinical pH-effect for new molecular entities that integrates physicochemical analysis and in vitro, in vivo and in silico methods.

The synthesis and SAR of calcitonin gene-related peptide (CGRP) receptor antagonists derived from tyrosine surrogates. Part 2.

Various substituted indazole and benzoxazolone amino acids were investigated as d-tyrosine surrogates in highly potent CGRP receptor antagonists. Compound 3, derived from the 7-methylindazole core, afforded a 30-fold increase in CGRP binding potency compared with its unsubstituted indazole analog 1. When dosed at 0.03mg/kg SC, compound 2 (a racemic mixture of 3 and its (S)-enantiomer) demonstrated robust inhibition of CGRP-induced increases in mamoset facial blood flow up to 105min. The compound possesses a favorable predictive in vitro toxicology profile, and good aqueous solubility. When dosed as a nasal spray in rabbits, 3 was rapidly absorbed and showed good intranasal bioavailability (42%).

Discovery of (R)-N-(3-(7-methyl-1H-indazol-5-yl)-1-(4-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide (BMS-742413): a potent human CGRP antagonist with superior safety profile for the treatment of migraine through intranasal delivery.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.

Strategies for Managing Solid Form Transformation Risk in Drug Product.

The International Conference of Harmonization (ICH) Q6A document provides guidance on setting specifications for new drug substances and drug products.1 In this paper we focus on decision trees 4 (#1) to (#3) in the guidance related to solid-state form transformation. Form transformation could occur from use of high energy forms to overcome solubility challenges or stresses from manufacturing processes. The decision trees provide guidance on when and how polymorphic form changes should be monitored and controlled. However, guidance is high level and does not capture aspects related to assessments needed to understand if there is a risk of transformation or tools that can be integrated to understand the severity of bioavailability impact at different stages of development. The objective of this paper is therefore to provide comprehensive chemistry manufacturing and controls (CMC) and regulatory strategies to manage the risk of form transformation. This includes practical workflows for form transformation risk assessment, analytical tools to detect and quantify the transformation including their shortcomings, biopharmaceutical tools to understand the severity of transformation risk and if needed justify the limits based on clinical relevance. Finally, a few case studies are discussed that capture how the workflow can be used to manage transformation risk.

Solution behavior of PVP-VA and HPMC-AS-based amorphous solid dispersions and their bioavailability implications.

PURPOSE: To identify the mechanism behind the unexpected bio-performance of two amorphous solid dispersions: BMS-A/PVP-VA and BMS-A/HPMC-AS. METHODS: Solubility of crystalline BMS-A in PVP-VA and HPMC-AS was measured by DSC. Drug-polymer interaction parameters were obtained by Flory-Huggins model fitting. Drug dissolution kinetics of spray-dried dispersions were studied under sink and non-sink conditions. BMS-A supersaturation was studied in the presence of pre-dissolved PVP-VA and HPMC-AS. Potency and crystallinity of undissolved solid dispersions were determined by HPLC and DSC. Polymer dissolution kinetics were obtained by mass balance calculation. Bioavailability of solid dispersions was assessed in dogs. RESULTS: In solid state, both polymers are miscible with BMS-A, while PVP-VA solublizes the drug better. BMS-A dissolves similarly from both solid dispersions in vitro regardless of dissolution method, while the HPMC-AS dispersion performed much better in vivo. At the same concentration, HPMC-AS is more effective in prolonging BMS-A supersaturation; this effect was negated by the slow dissolution rate of HPMC-AS. Further study revealed that fast PVP-VA dissolution resulted in elevated drug loading in undissolved dispersions and facilitated drug recrystallization before complete release. In contrast, the hydrophobicity and slower HPMC-AS dissolution prevented BMS-A recrystallization within the HPMC-AS matrix for >24 h. CONCLUSIONS: The lower bioavailability of PVP-VA dispersion was attributed to BMS-A recrystallization within the undissolved dispersion, due to hydrophilicity and fast PVP-VA dissolution rate. Polymer selection for solid dispersion development has significant impact on in vivo performance besides physical stability.

Pulmonary delivery of a GLP-1 receptor agonist, BMS-686117.

Alternate delivery route of therapeutic peptides is an attractive non-invasive option to patients who must chronically self-administer their medication through injections. In recent years, much attention has centered on pulmonary peptide delivery of peptide drugs such as insulin and GLP-1 mimetic peptides in the treatment of type II diabetes. In this study, we assessed the feasibility of delivering BMS-686117, an 11-mer GLP-1 receptor peptide agonist, to the lung in rats via intratracheal administration. The pharmacokinetic profiles of three spray-dried, prototype inhaled powder formulations, 80/20 BMS-686117/trehalose (I), 100% BMS-686117 (II), and 20/80 BMS-686117/mannitol (III), as well as a lyophilized BMS-686117 powder, were compared with intravenously and subcutaneously administered peptide. The spray-dried formulations were mostly spherical particles with narrow particle size distribution between 2 to 10 microm, which are better suited for inhalation delivery than the lyophilized, irregular shape powder with a wide particle size distribution between 2 to 100 microm. Prototype III exhibited the best physical characteristics and in vivo performance, with bioavailability of 45% relative to subcutaneous administration. The T(max) for lung delivered peptide formulations were almost twice as fast as subcutaneous injection, suggesting potential for rapid absorption and onset of action. This study demonstrated that pulmonary delivery is a promising, non-invasive route for the administration of BMS-686117.

Modulation of tight junctions does not predict oral absorption of hydrophilic compounds: use of Caco-2 and Calu-3 cells.

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.

Biopharmaceutical Evaluation and CMC Aspects of Oral Modified Release Formulations.

This article discusses the range of outcomes from biopharmaceutical studies of specific modified release (MR) product examples in preclinical models and humans. It touches upon five major biopharmaceutical areas for MR drug products: (1) evidence for regional permeability throughout the GI tract, (2) susceptibility to food-effect, (3) susceptibility to pH-effect, (4) impact of chronopharmacology in designing MR products, and (5) implications to narrow therapeutic index products. Robust bioperformance requires that product quality is met through a thorough understanding of the appropriate critical quality attributes that ensure reliable and robust manufacture of a MR dosage form. The quality-by-design (QbD) aspects of MR dosage form design and development are discussed with the emphasis on the regulatory view of the data required to support dosage form development.

Carbopol-mediated paracellular transport enhancement in Calu-3 cell layers.

The interaction of Carbopol polymers with mucus producing Calu-3 human bronchial epithelial cells was evaluated to test for potential paracellular transport enhancement. Using desmopressin (1-deamino-8-arginine-vasopressin, DDAVP) as the model peptide, apical treatment with Carbopol polymer gel formulations resulted in molecular size-dependent permeability enhancement with a concomitant drop in the transepithelial electrical resistance (TEER). Permeability enhancement of DDAVP was dependent on the formulation vehicle composition and polymer concentration, was noncytotoxic, and completely reversible. Carbopol 971P displayed the greatest permeability enhancement across Calu-3 cells compared to other more viscous Carbopol polymers 934P and 974P, and other mucoadhesive cellulosic polymers. The greatest enhancement was observed when C971P formulation was prepared in water at a concentration of 0.25% w/v. Enhancement was confirmed in rabbit dosed with intranasal fluorescent dextran 4400. The C(max) and absorption rate each increased by 48% in C971P formulations compared to control, while the relative exposure increased 30%. In conclusion, Carbopol polymers are potentially useful excipients to enhance intranasal peptide absorption. We hypothesize that the permeation enhancement is related to the chelation of extracellular or tight-junctional Ca(2+) by charged polymer carboxylate groups that leads to temporary disruption of tight-junctions, thereby facilitating paracellular transport.

A rabbit model for sublingual drug delivery: comparison with human pharmacokinetic studies of propranolol, verapamil and captopril.

A rabbit model for investigating sublingual drug absorption was established yielding results consistent with clinical data reported in the literature. Using propranolol as a model compound the effect of formulation and dosing variables was explored as a means to characterize the limiting parameters of this model. In addition, verapamil and captopril were selected as reference compounds to compare this model to sublingual absorption in humans. Rabbits were dosed sublingually and systemic absorption was measured over time. Sublingual absorption of propranolol was dependent on dosing solution pH and volume. Intra-oral spray device did not affect the overall exposure compared to instillation using a syringe. Despite species and dosing regimen differences the relative bioavailabilities of propranolol and verapamil were very similar in rabbits and humans. In contrast, captopril absorption from the sublingual cavity of rabbits was low and did not agree with that observed in man. Here we report a sublingual rabbit model of drug delivery and its potential utility in preclinical development of intra-oral dosage forms.

Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells.

The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.

Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.

Food Effect in Humans: Predicting the Risk Through In Vitro Dissolution and In Vivo Pharmacokinetic Models.

In vitro and in vivo experimental models are frequently used to assess a new chemical entity's (NCE) biopharmaceutical performance risk for food effect (FE) in humans. Their ability to predict human FE hinges on replicating key features of clinical FE studies and building an in vitro-in vivo relationship (IVIVR). In this study, 22 compounds that span a wide range of physicochemical properties, Biopharmaceutics Classification System (BCS) classes, and food sensitivity were evaluated for biorelevant dissolution in fasted- and fed-state intestinal media and the dog fed/fasted-state pharmacokinetic model. Using the area under the curve (AUC) as a performance measure, the ratio of the fed-to-fasted AUC (FE ratio) was used to correlate each experimental model to FE ratio in humans. A linear correlation was observed for the in vitro dissolution-human IVIVR (R (2) = 0.66, % mean square error 20.7%). Similarly, the dog FE ratio correlated linearly with the FE ratio in humans (R (2) = 0.74, % mean square error 16.25%) for 15 compounds. Data points near the correlation line indicate dissolution-driven mechanism for food effect, while deviations from the correlation line shed light on unique mechanisms that can come into play such as GI physiology or unusual physicochemical properties. In summary, fed/fasted dissolution studies and dog PK studies show a reasonable correlation to human FE, hence are useful tools to flag high-risk NCEs entering clinical development. Combining kinetic dissolution, dog FE model and in silico modeling one can study FE mechanism and formulation strategies to mitigate the FE risk.

Effect of delivery route on pulmonary response to oncostatin M.

The effect of delivery route on lung tissue response to oncostatin M (OM) was evaluated in isolated perfused rat lung (IPRL) and normal human bronchial epithelial cell line BEAS-2B in vitro models. In this study, the extent of induction of the cytokine IL-6 by OM was examined as evidence for local pharmacological activity in response to OM in lung tissue. OM stimulated dose-dependent release of IL-6 in both BEAS-2B cells and IPRL after exposure of either the apical (AP) or basolateral (BL) side of the epithelium. The increase in IL-6 was rapid, beginning 2 h after dosing, and sustained over 48 h. Similar amounts of IL-6 were released to both AP and BL sides of BEAS-2B cells, regardless of the side of OM dosing. However, in IPRL the extent of response of IL-6 induction to OM was always higher at the dosing side. In addition, airway dosing of OM resulted in an overall lower IL-6 response than perfusate dosing, which may result from the low rate of OM transport (<1%/h) across pulmonary epithelium. We conclude that the route of delivery significantly affects the overall extent of the pharmacological response to OM, with a lesser and more localized response after airway delivery to the lung.