Characterization of a novel non-steroidal glucocorticoid receptor agonist optimized for topical treatment.

Glucocorticoids (GCs) are commonly used topical treatments for skin diseases but are associated with both local and systemic side effects. In this study, we describe a selective non-steroidal glucocorticoid receptor (GR) agonist for topical use, LEO 134310, which is rapidly deactivated in the blood resulting in low systemic exposure and a higher therapeutic index in the TPA-induced skin inflammation mouse model compared with betamethasone valerate (BMV) and clobetasol propionate (CP). Selectivity of LEO 134310 for GR was confirmed within a panel of nuclear receptors, including the mineralocorticoid receptor (MR), which has been associated with induction of skin atrophy. Topical treatment with LEO 134310 in minipigs did not result in any significant reduction in epidermal thickness in contrast to significant epidermal thinning induced by treatment with BMV and CP. Thus, the profile of LEO 134310 may potentially provide an effective and safer treatment option for skin diseases compared with currently used glucocorticoids.

Characterizing Cutaneous Drug Delivery Using Open-Flow Microperfusion and Mass Spectrometry Imaging.

Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.

Label-Free Quantification of Pharmacokinetics in Skin with Stimulated Raman Scattering Microscopy and Deep Learning.

The treatment of inflammatory skin conditions relies on a deep understanding of how drugs and tissue behave and interact. Although numerous methods have been developed that aim to follow and quantify topical drug pharmacokinetics, these tools can come with limitations, assumptions, and trade-offs that do not allow for real-time tracking of drug flow and flux on the cellular level in situ. We have developed a quantitative imaging toolkit that makes use of stimulated Raman scattering microscopy and deep learning-based computational image analysis to quantify the uptake of specific drug molecules in skin without the need for labels. Analysis powered by trained convolutional neural networks precisely identified features such as cells, cell junctions, and cell types within skin to enable multifactorial analysis of skin pharmacokinetics. We imaged and quantified the flow and flux of small molecule drugs through the layers and structures of ex vivo nude mouse ear skin and extracted pharmacokinetic parameters through convolutional neural network-based image processing, including relative area under the curve accumulation, time of maximum drug concentration, and in situ partition ratios. This approach, which facilitates the direct observation and quantification of pharmacokinetics, can be used to glean mechanistic insight into underlying phenomena in skin pharmacokinetics.

Evaluating Dermal Pharmacokinetics and Pharmacodymanic Effect of Soft Topical PDE4 Inhibitors: Open Flow Microperfusion and Skin Biopsies.

PURPOSE: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants. METHODS: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement. RESULTS: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations. CONCLUSIONS: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

Discovery and Early Clinical Development of Isobutyl 1-[8-Methoxy-5-(1-oxo-3H-isobenzofuran-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylate (LEO 39652), a Novel "Dual-Soft" PDE4 Inhibitor for Topical Treatment of Atopic Dermatitis.

We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while being stable in human skin. Compound 40 was selected as a clinical candidate as it was potent and rapidly degraded by blood and liver to inactive metabolites and because in preclinical studies it showed high exposure at the target organ: the skin. Preclinical and clinical data are presented confirming the value of the dual-soft concept in reducing systemic exposure.

Topical 'dual-soft' glucocorticoid receptor agonist for dermatology.

Steroidal glucocorticoids (GR agonists) have been widely used for the topical treatment of skin disorders, including atopic dermatitis. They are a very effective therapy, but they are associated with both unwanted local effects in the skin (skin thinning/atrophy) and systemic side effects. These effects can limit the long-term utility of potent steroids. Here we report on a topically delivered non-steroidal GR agonist, that has the potential to deliver high efficacy in the skin, but due to rapid metabolism in the blood & liver ("dual-soft") it should have greater systemic safety than existing treatments. In addition, compared to less selective steroidal GR agonists, the new non-steroidal Selective Glucocorticoid Agonists (SEGRAs) have the potential to avoid the skin atrophy observed with existing topical steroids. Due to its potential for reduced skin atrophy and low systemic exposure, LEO 134310 (17) may be suitable for long term topical treatment of skin diseases such as atopic dermatitis and psoriasis.

Role of Substance P and Its Receptor Neurokinin 1 in Chronic Prurigo: A Randomized, Proof-of-Concept, Controlled Trial with Topical Aprepitant.

Substance P (SP) and its receptor neurokinin 1 (NK1R) are thought to be involved in the pathogenesis of chronic prurigo. Here, we assessed SP serum levels, cutaneous NK1R expression, and the effects of topical aprepitant, an NK1R antagonist, in patients with chronic prurigo. SP and NK1R were increased, compared with controls, in the serum and in lesional vs. non-lesional skin of the patients, respectively. Aprepitant, in a randomized, placebo-controlled, split-sided, doubleblind trial, reduced the intensity of pruritus as assessed by visual analogue scale by >50% from baseline to day 28 (-35.2), but so did placebo vehicle (-38.1, p= 0.76). Overall clinical scores improved significantly by day 28 in both treatment groups, with no significant difference between the 2 groups (p=0.32). Our findings imply that both SP and NK1R are involved in the pathogenesis of chronic prurigo. Parallel groupdesigned trials are needed to assess the efficacy of topical aprepitant treatment in this condition.

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

DissolvIt: An In Vitro Method for Simulating the Dissolution and Absorption of Inhaled Dry Powder Drugs in the Lungs.

The main purpose of this work was to develop an in vitro method for simulating the dissolution and absorption of inhaled dry powder drugs that also mimics systemic pharmacokinetic data. A second purpose was to evaluate this method. DissolvIt® was developed as a simulation of the air-blood barrier of the upper airways, constituting: "airborne" particles deposited on a glass cover slip, a mucus simulant, a polycarbonate (basal) membrane, and a pumped albumin buffer simulating the pulmonary blood flow. The PreciseInhale® exposure system was used to aerosolize and deposit test formulations onto cover slips. The particle dissolution was observed by optical microscopy as particle disappearance, and it was started directly when the particles came into contact with the mucus simulant. Solute from the dissolving particles diffused through the barrier and was absorbed into the perfusate. The drug concentration in the perfusate over time and the remaining drug in the barrier at the end of the experiment were quantitated by using liquid chromatography-tandem mass spectrometry. Budesonide and fluticasone propionate generated different pharmacokinetic dissolution/absorption profiles in DissolvIt. This study indicates that DissolvIt simulates dissolution and absorption of drugs in the lung, and that DissolvIt also mimics pharmacokinetic profiles and parameters.

Imaging of distribution of topically applied drug molecules in mouse skin by combination of time-of-flight secondary ion mass spectrometry and scanning electron microscopy.

In the development of topical drugs intended for local effects in the skin, one of the major challenges is to achieve drug penetration through the external barrier of the skin, stratum corneum, and secure exposure to the viable skin layers. Mass spectrometric imaging offers an opportunity to study drug penetration in a variety of skin models by mapping the spatial distribution in different skin layers after topical application of the drug. In this study, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) and scanning electron microscopy (SEM) to image the distribution of three drug molecules in skin tissue cross sections of inflamed mouse ear. The three compounds, roflumilast, tofacitinib, and ruxolitinib, were topically administered to the mouse ears, which were subsequently cryosectioned and thawed for the analyses. The results reveal that the combination of TOF-SIMS and SEM was beneficial for interpretation of drug distribution. SEM identified the different skin layers, while spatial distributions of all three compounds could be visualized by TOF-SIMS, showing that the drug was primarily distributed into, or on the top of, the stratum corneum. Imaging of endogenous skin components like cholesterol, phospholipids, ceramides, and free fatty acids showed distributions in good agreement with the literature. One limitation of the TOF-SIMS method is sensitivity, typically allowing for analysis in the millimolar range rather than the pharmacologically relevant micromolar range. However, the data presented demonstrate the potential of the technique for studying the penetration of drugs with different physicochemical properties in skin.

Combined budesonide/formoterol therapy in conjunction with allergen avoidance ameliorates house dust mite-induced airway remodeling and dysfunction.

Allergic asthma is characterized by airway inflammation in response to chronic allergen exposure, resulting in remodeling of the airway wall accompanied by dysfunctional airway physiology. However, a link between the immune-inflammatory response to allergen and changes to airway structure and physiology has not yet been fully elucidated. Moreover, the impact of inhaled corticosteroids and beta(2)-agonists, the primary pharmacotherapy for asthma, on this process has not been completely evaluated. In this study, we employed a murine model of chronic exposure to a common environmental aeroallergen, house dust mite, to recapitulate the phenotype of clinical asthma. By examining the therapeutic effects of corticosteroid/beta(2)-agonist combination therapy with budesonide/formoterol (BUD/FORM) in this model of airway disease, we endeavored to determine the impact of BUD/FORM on lung inflammation, structure, and physiology. BUD/FORM was delivered either while allergen exposure was ongoing (concurrent therapy) or following the cessation of allergen exposure (postexposure therapy). Our results show that airway inflammation was substantially reduced in BUD/FORM-treated mice in the concurrent therapy group, whereas in the postexposure therapy group airway inflammation spontaneously resolved. In contrast, BUD/FORM was most effective in resolving several aspects of airway remodeling and bronchial hyperreactivity when delivered in conjunction with allergen withdrawal. This study demonstrates that although both BUD/FORM therapy and allergen avoidance independently reduce airway inflammation, only BUD/FORM therapy in conjunction with allergen avoidance can effectively reverse airway remodeling and bronchial hyperreactivity induced by chronic allergen exposure.

Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol, and terbutaline.

There is an increasing interest in using the lung as a route of entry for both local and systemic administration of drugs. However, because adequate technologies have been missing in the preclinical setting, few investigators have addressed the detailed disposition of drugs in the lung following short inhalation exposures to highly concentrated dry powder aerosols. New methods are needed to explore the disposition of drugs after short inhalation exposures, thus mimicking a future clinical use. Our aim was to study the pulmonary disposition of budesonide, formoterol, and terbutaline, which are clinically used for the treatment of bronchial asthma. Using the recently developed DustGun aerosol technology, we exposed by inhalation for approximately 1 min the isolated and perfused rat lung (IPL) to respirable dry particle aerosols of the three drugs at high concentrations. The typical aerosol concentration was 1 mug/mL, and the particle size distribution of the tested substances varied with a MMAD ranging from 2.3 to 5.3 mum. The IPL was perfused in single pass mode and repeated samples of the perfusate were taken for up to 80 min postexposure. The concentration of drug in perfusate and in lung extracts was measured using LC-MS/MS. The deposited dose was determined by adding the amounts of drug collected in perfusate to the amount extracted from the tissues at 80 min. Deposited amounts of budesonide, formoterol fumarate, and terbutaline sulphate were 23 +/- 17, 36 +/- 8, and 60 +/- 3.2 mug (mean +/- SD, n = 3), respectively. Retention in lung tissues at the end of the perfusion period expressed as fraction of deposited dose was 0.19 +/- 0.05, 0.19 +/- 0.06, and 0.04 +/- 0.01 (mean +/- SD, n = 3) for budesonide, formoterol, and terbutaline, respectively. Each short inhalation exposure to the highly concentrated aerosols consumed 1-3 mg powder. Hence, this system can be particularly useful for obtaining a detailed pharmacokinetic characterization of inhaled compounds in drug discovery/development.

An optimized automated assay for determination of metabolic stability using hepatocytes: assay validation, variance component analysis, and in vivo relevance.

Screening of new chemical entities for metabolic stability using hepatocytes is routinely used in drug discovery. To enhance compound throughput, an optimized automated microassay for determination of intrinsic clearance was developed. Dulbecco's modified Eagle's medium, Hanks' balanced salt solution, and Leibovitz L-15 medium (L-15) were tested for their ability to maintain cell viability during incubation in 96-well plates. L-15 was found to keep pH within 0.1 units and maintain high viability during several hours of incubation. Moreover, two different thawing protocols for cryopreserved hepatocytes were compared. Protocol 2 resulted in a nearly 100% increase in post-thaw yield, whereas no difference was observed in cell viability. The microassay was validated using human cryopreserved hepatocytes and 19 reference compounds covering the most important phase I and II liver metabolizing enzymes ranging from low to medium and high clearance compounds. The day-to-day variation was determined, revealing an overall good precision of the assay. In vitro-in vivo correlations, for both fresh rat and cryopreserved human hepatocytes, were calculated. For 86% (human) and 77% (rat) of the compounds, calculated hepatic clearance was within twofold observed clearance in vivo. Using the validation data, variance component analysis was applied to determine within and between-experiment variability, enabling estimation of variation and detection limit for any combination of repeated experiments and replicate samples. Based on the precision desired, this provides a tool to select the most optimal and cost-effective assay approach for different compounds considering the actual phase in the drug discovery program.

Miniaturized nebulization catheters: a new approach for delivery of defined aerosol doses to the rat lung.

A nebulization catheter technique (AeroProbe) was adapted and evaluated as a new approach for pulmonary delivery of defined aerosol doses to the rat lung. The lung distribution profile was evaluated by dosing Evans blue and Nile blue dye, respectively, to isolated and perfused rat lungs (IPL) and to the lungs of anesthetized and tracheal-intubated rats. The intratracheal aerosol dosing was synchronized with the inspiration of the lungs. Immediately after dosing, the lungs were dissected into upper- and lower trachea, bronchi, and parenchyma. The dye was then extracted from the tissue samples to determine the regional distribution and the recovery of the aerosol dose in the lungs. The droplet-size distribution and the weight of the delivered aerosol dose were analyzed with laser diffraction and gravimetric analysis respectively. The recovery of the delivered dose was high, 99 +/- 12 and 105 +/- 1%, respectively, in the in vivo administrations and IPL-experiments. The lung distribution profile after aerosol dosing to anesthetized rats was mainly tracheobronchial. Only 12 +/- 4% of the dose was recovered in the lung parenchyma. However, after aerosol dosing to the IPL, 38 +/- 11% of the dose was distributed to the lung parenchyma. At the settings used, the nebulization catheter aerosolized 1-2 microL of liquid per puff using 1-1.5 mL of air. The droplet-size distribution of the generated aerosols was broad (2-8% <3 microm; 10% <4-7 microm; 50% <10-15 microm; and 90% <20-40 microm). The nebulization catheter technique provides a complement to existing methodology for pulmonary drug delivery in small animals. With this new technique, defined aerosol doses can be delivered into the lungs of rats with no need for aerosol dosimetry.

Mechanisms for absorption enhancement of inhaled insulin by sodium taurocholate.

The effects of sodium taurocholate (NaTC) on the absorption of inhaled insulin was investigated using both in vivo and in vitro experiments. The absolute bioavailability of insulin when given as a nebulized solution (0.6 mM) to anesthetized intubated beagle dogs was low (2.6+/-0.3%). However, when NaTC at different concentrations (2-32 mM) were included in the formulations the bioavailability increased and at 32 mM it was about nine times higher (23.2+/-4.4%) than for pure insulin. In a similar concentration interval (20-25 mM) NaTC decreased the transepithelial electrical resistance (TEER) across Caco-2 cell monolayers leading to an increased permeability of insulin. At higher concentrations (above 30 mM) the viability of the Caco-2 cells decreased and the insulin permeability increased dramatically. Furthermore, we show that NaTC in the concentration range 2-15 mM gradually decreases the aggregation state of insulin, i.e., produces mono- or dimeric insulin from hexameric insulin. In conclusion, NaTC increases the bioavailability of nebulized insulin, increases the permeability of insulin across Caco-2 cell monolayers, and decreases the aggregation state of insulin at similar concentrations. We suggest that the main mechanisms behind the absorption enhancement of inhaled insulin by NaTC are the production of insulin monomers and an opening of tight junctions between adjacent airway epithelial cells.

Enhanced insulin absorption in the rabbit airways and lung by sodium dioctyl sulfosuccinate.

The objective of this investigation was to study regional absorption of inhaled insulin together with an enhancer (sodium di-octyl-sulfosuccinate [DOSS]) in the rabbit airways and lung. Insulin was administered with or without DOSS by aerosol inhalation, intratracheal infusion, intranasally, sublingually, and without DOSS intravenously. Blood glucose and plasma levels of insulin were measured during 100 min from the start of administration. Inhalation of insulin (3 U) with 0.25% or 1% DOSS decreased average blood glucose levels significantly more than inhalation of insulin (3 U) without DOSS. Intratracheal administration of 1.5 U of insulin with 0.25% DOSS in 0.3 mL of vehicle decreased the average blood glucose level significantly compared with intratracheal administration of 1.5 U of insulin and no DOSS in 0.3 mL of vehicle and compared with 1.5 U of insulin with 0.25% DOSS in 0.15 mL of vehicle. Intravenous insulin (1.5 U) and inhaled (1.5 U) insulin in 0.25% DOSS decreased average blood glucose levels significantly compared with intratracheal (0.15 mL), intranasal, and sublingual administration of 1.5 U of insulin with 0.25% DOSS. The bioavailability of inhaled insulin (1.5 U) with 0.25% DOSS was estimated to be 16% in comparison with 7% for intratracheally (0.15 mL), 1% intranasally, and 0.8% sublingually administered insulin (1.5 U with 0.25% DOSS), respectively. Inhaled insulin together with the absorption enhancer DOSS decreased the blood glucose level more effectively than insulin given intratracheally, intranasally, or sublingually. The effect on blood glucose reflected the difference in plasma insulin concentration for the different routes of administration.

Regional differences in bioavailability of an opioid tetrapeptide in vivo in rats after administration to the respiratory tract.

TArPP (Tyr-D-Arg-Phe-Phe-NH(2)), 1-10 micromol/kg, was administered to anesthetized rats by nasal microinfusion, intratracheal microinfusion, intratracheal nebulization, aerosol inhalation, and i.v. bolus and infusion. Plasma concentrations of TArPP and its deamidated metabolite were determined by LC-MS-MS. Regional differences in bioavailability (F), first-pass metabolism, and absorption rate were found for TArPP after delivery to the respiratory tract. Absorption was rapid after both pulmonary and nasal administration (t(max) approximately 10-20 min). After nasal microinfusion, F was 52 +/- 9%. For all the pulmonary groups, F was higher (72-114%). First-pass metabolism of TArPP was lower in the lung than in the nasal cavity. It is evident that the pulmonary route is attractive for successful systemic delivery of small, hydrophilic and enzymatic susceptible peptides.