Anne Cooper

Optimisation of DMPK by the inhaled route: challenges and approaches.

The renewed interest in inhalation delivery over recent years has led to an expansion in the understanding of lung pharmacokinetics. Historically optimisation of inhaled drugs focused largely on development of material properties, consistent with achieving a good lung deposition, alongside demonstrating appropriate in vivo efficacy with little understanding of the relationship to pharmacokinetics in the lung. Recent efforts have led to an increased understanding of lung concentrations and how to maximise exposure in order to achieve the desired pharmacological response at a dose consistent with development of an inhaled product. Although there is a prerequisite for excellent potency in inhalation delivery, it is essential that this be combined with pharmacokinetic properties that allow sufficient free concentration at the effect site in lung to exert the pharmacological response for an appropriate dosing interval. Increases in basicity, polarity and/or decreases in aqueous solubility can extend pharmacokinetic duration and assist in finding the right balance between lung and systemic exposure. Current evidence suggests there are similarities in lung retention in rat and dog and that animal lung concentration data can enable pharmacokinetic-pharmacodynamic relationships to be derived thus providing more confidence in the requirements for man. Although inhaled delivery is challenging from a pharmacokinetic point of view, direct evaluation of exposure in the target organ has enabled further understanding of the drivers for drug disposition and highlighted the need for further development of predictive lung pharmacokinetic tools in the future.

Hit-to-lead studies: the discovery of potent, orally bioavailable triazolethiol CXCR2 receptor antagonists.

A Hit-to-Lead optimisation programme was carried out on the high throughput screening hit, the triazolethiol 1, resulting in the discovery of the potent, orally bioavailable triazolethiol CXCR2 receptor antagonist 45.

P-glycoprotein Potentiates CYP3A4-mediated Drug Disappearance during Caco-2 Intestinal Secretory Detoxification

Human intestinal Caco-2 cell monolayers grown in the presence of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) were used to test the hypothesis that drugs which interact with the apical efflux pump P-glycoprotein (Pgp) may enhance CYP3A4-mediated disappearance of substrates. 6β-Hydroxytestosterone production, a marker of CYP3A4 activity, was approximately 3- and 7-fold greater in 1,25(OH)2D3-treated cells compared to untreated cells when incubated with 50 and 500 μM testosterone, respectively, and was unaffected by the addition of digoxin to reduce Pgp activity. In the presence of digoxin, secretory transport of vinblastine and erythromycin, substrates for both Pgp and cytochrome P450 3A4 (CYP3A4), was significantly reduced, whereas absorptive transport was unaffected. In contrast, no directional transport of testosterone, a substrate for CYP3A4 only, was observed, either in the presence or absence of digoxin. Over 2 h, disappearance of erythromycin and vinblastine from the incubation medium was significantly greater from the basolateral than from the apical compartments. In the presence of digoxin, disappearance of both compounds from the basolateral, but not from the apical compartments, was significantly reduced. In contrast, disappearance of testosterone was unaffected by the addition of digoxin, demonstrating that the effect of digoxin on erythromycin and vinblastine disappearance was via inhibition of Pgp function, rather than on CYP3A4 activity. Thus, evidence is provided for Pgp/CYP3A4 co-substrates, Pgp potentiates CYP3A4-mediated drug disappearance during intestinal secretory detoxification.

Prediction of Efficacious Inhalation Lung Doses via the Use of In Silico Lung Retention Quantitative Structure-Activity Relationship Models and In Vitro Potency Screens

Lung concentrations of a drug are expected to drive the pharmacodynamic response to local inflammation after inhalation delivery, and the only way of determining the efficacious dose has been to measure it directly in animal models. In this study, we present a method to predict efficacious lung doses after inhalation in a rat lipopolysaccharide challenge model from in silico predictions of lung concentration and in vitro measurements only. A quantitative structure-activity relationship (QSAR) model, based on calculated physical properties predicted the partitioning of 34 compounds between lung and plasma. Because it was observed that lung/plasma partitioning correlated with lung concentration, it was possible to use this relationship to predict lung concentration at a given dose and time point. Based on the pharmacokinetic-pharmacodynamic (PKPD) relationship observed, a minimal free lung concentration relative to potency to drive significant inhibition of neutrophilia was established. By using predicted lung concentrations, measured fraction unbound in plasma, and cellular potency, it was possible to estimate an inhaled lung dose that would be expected to achieve this target exposure. These predictions were made for 23 compounds, which were not part of the original QSAR training set, and all except one were predicted to within 3-fold of their measured values. This novel approach shows that by understanding PKPD relationships and drivers for lung affinity after inhalation dosing, it is possible to estimate in vivo lung doses required for efficacy. This methodology provides a useful screening tool to rank candidate compounds and minimizes the use of extensive animal testing.

The Discovery of Novel, Potent and Highly Selective Inhibitors of Inducible Nitric Oxide Synthase (Inos).

By careful analysis of experimental X-ray ligand crystallographic protein data across several inhibitor series we have discovered a novel, potent and selective series of iNOS inhibitors exemplified by compound 8.

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1.

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.

Unusual structure–genotoxicity relationship in mouse lymphoma cells observed with a series of kinase inhibitors

During development of a novel kinase inhibitor for an anti-inflammatory therapy at AstraZeneca UK, the lead compound was found to be potently active in the mouse lymphoma assay (MLA). This was not believed to be due to primary pharmacology because structural alert relationships and a negative Ames test indicated that the compound was unlikely to form DNA adducts. A number of investigations were performed to assess whether mammalian cell genotoxicity was inherent to the chemical series. The in vitro micronucleus assay (MN(vit)) combined with a semi-automated analysis system, was used as a high-throughput screen. A number of additional compounds were selected for testing, all with different substituents around a core isoquinolinone. These modifications did not affect the kinase and non-kinase selectivity of the compounds. Several of these compounds were positive in the MN(vit), however, two compounds were found to be negative and these were also confirmed to be negative in the MLA. It was considered possible that topoisomerase II or off-target kinase inhibition may have been responsible for the observed mammalian cell genotoxicity. The present investigations show how an iterative chemical design, along with genotoxicity screening by use of a semi-automated MN(vit), can identify and remove the genotoxic hazard from pharmaceutical projects at an early stage of development, and produce high-quality molecules suitable for further progression.

Evaluation of layers of the rat airway epithelial cell line RL-65 for permeability screening of inhaled drug candidates.

A rat respiratory epithelial cell culture system for in vitro prediction of drug pulmonary absorption is currently lacking. Such a model may however enhance the understanding of interspecies differences in inhaled drug pharmacokinetics by filling the gap between human in vitro and rat in/ex vivo drug permeability screens. The rat airway epithelial cell line RL-65 was cultured on Transwell inserts for up to 21 days at an air-liquid (AL) interface and cell layers were evaluated for their suitability as a drug permeability measurement tool. These layers were found to be morphologically representative of the bronchial/bronchiolar epithelium when cultured for 8 days in a defined serum-free medium. In addition, RL-65 layers developed epithelial barrier properties with a transepithelial electrical resistance (TEER) >300 Ω cm(2) and apparent (14)C-mannitol permeability (P(app)) values between 0.5-3.0 × 10(-6)cm/s; i.e., in the same range as established in vitro human bronchial epithelial absorption models. Expression of P-glycoprotein was confirmed by gene analysis and immunohistochemistry. Nevertheless, no vectorial transport of the established substrates (3)H-digoxin and Rhodamine123 was observed across the layers. Although preliminary, this study shows RL-65 cell layers have the potential to become a useful in vitro screening tool in the pre-clinical development of inhaled drug candidates.