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Magnus Nilsson,1 Magdalena Rhedin,2 Ramon Hendrickx,3 Susanne Berglund,1 Antonio Piras,2 Parmis Blomgran,2 Anders Cavallin,2 Mia Collins,2 Göran Dahl,4 Bilel Dekkak,5 Therese Ericsson,3 Niklas Hagberg,6 Ann Aurell Holmberg,3 Agnes Leffler,2 Anders J Lundqvist,3 Thomais Markou,5,7 James Pinkerton,5,7 Lars Rönnblom,6 Stacey Siu,8 Vanessa Taylor,8 Tiiu Wennberg,2 Dimitrios Zervas,5,7 Arian D J Laurence,9 Suman Mitra,2 Maria G Belvisi,5,7 Mark Birrell,5,7 Annika Borde2 1Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 2Bioscience, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 3DMPK, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Discovery Science, R&D, AstraZeneca, Gothenburg, Sweden; 5Respiratory Pharmacology Group, Division of Airway Disease, National Heart and Lung Institute, Imperial College London, London, UK; 6Rheumatology and Science for Life Laboratories, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; 7Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 8Rigel Pharmaceuticals, South San Francisco, CA, USA; 9Department of Haematology, University College London Hospitals NHS Foundation Trust, London, UKCorrespondence: Magnus Nilsson, Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, SE-431 83, Sweden, Tel +46722237222, Email Magnus.Nilsson@astrazeneca.comPurpose: Janus kinase 1 (JAK1) is implicated in multiple inflammatory pathways that are critical for the pathogenesis of asthma, including the interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin cytokine signaling pathways, which have previously been targeted to treat allergic asthma. Here, we describe the development of AZD0449 and AZD4604, two novel and highly selective JAK1 inhibitors with promising properties for inhalation.Methods: The effects of AZD0449 and AZD4604 in JAK1 signaling pathways were assessed by measuring phosphorylation of signal transducer and activator of transcription (STAT) proteins and chemokine release using immunoassays of whole blood from healthy human volunteers and rats. Pharmacokinetic studies performed on rats evaluated AZD0449 at a lung deposited dose of 52 μg/kg and AZD4604 at 30 μg/kg. The efficacy of AZD0449 and AZD4604 was assessed by evaluating lung inflammation (cell count and cytokine levels) and the late asthmatic response (average enhanced pause [Penh]).Results: Both compounds inhibited JAK1-dependent cytokine signaling pathways in a dose-dependent manner in human and rat leukocytes. After intratracheal administration in rats, both compounds exhibited low systemic exposures and medium-to-long terminal lung half-lives (AZD0449, 34 hours; AZD4604, 5 hours). Both compounds inhibited STAT3 and STAT5 phosphorylation in lung tissue from ovalbumin (OVA)-challenged rats. AZD0449 and AZD4604 also inhibited eosinophilia in the lung and reduced the late asthmatic response, measured as Penh in the OVA rat model.Conclusion: AZD0449 and AZD4604 show potential as inhibitors of signaling pathways involved in asthmatic immune responses, with target engagement demonstrated locally in the lung. These findings support the clinical development of AZD0449 and AZD4604 for the treatment of patients with asthma.Keywords: AZD0449, AZD4604, JAK, STAT

Introduction: Asthma is a chronic inflammatory disease of the airways with typical symptoms manifesting in coughing, wheezing and dyspnoea. Whilst current treatments can be effective, some patients are not properly controlled, and the medications are not ideal and are associated with unwanted side effects. Thus, new therapies are urgently required. Typically, the chronic inflammation endotype is associated with increased levels of type 2 cytokine such as IL-4, IL-5, IL-9 & IL-13. These pro-inflammatory signalling pathways critically depend on Janus Kinase (JAK) I or II signal transduction, therefore the inhibition of JAK pathways represents a promising therapeutic target in asthma. Aims and Objectives: To assess the potential benefit of JAK inhibition using two structurally distinct inhibitors in a in vivo model system. Methods: Two JAK inhibitors, AZD4604 & AZD0449, were intratracheally dosed into sensitised Brown Norway rats one hour prior to aerosolised allergen (OVA) challenge. In one set of animals we assessed target engagement (TE) in the lung tissue by measuring levels of pSTATs. In a parallel set, we measured the Late Asthmatic Response (LAR) and lung inflammation. Results: Administration of both AZD4604 and AZD0449 resulted in dose related reduction of pSTAT3 and pSTAT5, indicating TE in the lung. Furthermore, AZD4604 & AZD0449 administration suppressed LAR and airway inflammation to a similar level to that achieved with the ICS comparator, budesonide. Conclusions: These data indicate that JAK inhibition may be of benefit to patients suffering from allergic asthma.

Inhaled drugs generally aim to drive a local pharmacological effect in lung, at the same time minimizing systemic exposure, in order to obtain efficacy in lung disease without unwanted systemic effects. Here, we demonstrate that inhaled delivery of a p38 inhibitor (AZD7624) can provide superior pharmacokinetic exposure and superior pharmacodynamic lung effects. In rats, inhaled AZD7624 had a five times higher dose-adjusted lung exposure compared with intravenous dosing. In healthy volunteers, lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) in sputum has been shown to be significantly reduced (85%) by means of inhaled AZD7624. Here, we demonstrate that this effect is associated with a mean unbound plasma concentration, gained from in vitro and ex vivo LPS-challenge protocols, significantly below potencies obtained for AZD7624, suggesting that lung exposure is probably much higher than systemic exposure. This assessment was made for the unbound potency (pIC50u), e.g., the potency remaining after adjustment for plasma protein binding and blood plasma ratio. Hence, the unbound potency of AZD7624 to inhibit LPS-induced TNFα in human mononuclear cells, in whole blood as well as in alveolar macrophages in vitro, was 8.4, 8.7 (full inhibition), and 9.0 (partial inhibition), respectively. The pIC50u in whole blood ex vivo was 8.8, showing good in vitro/in vivo potency correlations. Thus, a mean unbound AZD7624 plasma concentration of 0.3 nmol/l, which was associated with a decrease in LPS-induced sputum TNFα by 85%, is much lower than the pIC50u. This demonstrates that AZD7624 can achieve significant local lung pharmacodynamic effects with concomitant sub-pharmacological systemic exposure.

This review aims to raise the potential of the modern society's impact on gut integrity often leading to increased intestinal permeability, as a cause or driver of Alopecia Areata (AA) in genetically susceptible people. With the increasing rate of T cell-driven autoimmunity, we hypothesize that there is a common root cause of these diseases that originates from chronic inflammation, and that the gut is the most commonly exposed area with our modern lifestyle. Areas covered: We will discuss the complexity in the induction of AA and its potential link to increased intestinal permeability. Our main focus will be on the gut microbiome and mechanisms involved in the interplay with the immune system that may lead to local and/or peripheral inflammation and finally, tissue destruction. Expert opinion: We have seen a link between AA and a dysfunctional gastrointestinal system which raised the hypothesis that an underlying intestinal inflammation drives the priming and dysregulation of immune cells that lead to hair follicle destruction. While it is still important to resolve local inflammation and restore the IP around the hair follicles, we believe that the root cause needs to be eradicated by long-term interventions to extinguish the fire driving the disease.

Inhaled drugs generally aim to drive a local pharmacological effect in lung, at the same time minimizing systemic exposure, in order to obtain efficacy in lung disease without unwanted systemic effects. Here, we demonstrate that inhaled delivery of a p38 inhibitor (AZD7624) can provide superior pharmacokinetic exposure and superior pharmacodynamic lung effects. In rats, inhaled AZD7624 had a five times higher dose-adjusted lung exposure compared with intravenous dosing. In healthy volunteers, lipopolysaccharide (LPS)-induced tumor necrosis factor

Different oral liquid cholera vaccines have proved to be safe and effective, but their formulations present problems for use in low-income countries, since large package volumes have to be transported and cold chain maintenance is required. A solid state formulation would here be more advantageous, and consequently, the possibility to develop a dry cholera vaccine formulation by freeze-drying was investigated. The ability of sucrose, trehalose and mannitol to provide process stabilization during freeze-drying was tested on a formalin-killed whole-cell Vibrio cholerae model vaccine. A matrix of sucrose or trehalose prevented bacterial aggregation, preserved cell morphology and maintained practically completely the protective lipopolysaccharide (LPS) antigen on the cell surface and its reactivity with specific antibody in vitro. After reconstitution, this formulation also retained the capacity to elicit a strong serum and gut mucosal anti-LPS antibody response in orally immunized mice, as compared to the corresponding liquid vaccine formulation. The full preservation of the in vivo immunogenicity was also maintained when the internationally widely licensed oral cholera vaccine Dukoral™, which comprises a cocktail of inactivated V. cholerae together with cholera toxin B-subunit (CTB), was freeze-dried using sucrose for stabilization. Thus, we present a process generating a dry oral inactivated whole-cell cholera vaccine formulation with attractive features for public health use in cholera-afflicted settings.

A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation.

Carboxymethyl‐hexanoyl chitosan (CHC) has the ability to self‐assemble into nanocapsules in an aqueous solution and it has recently shown potential in numerous biomedical applications. Here we investigate the protein loading efficiency and release, as well as the structural properties of CHC protein nanocarriers. Bovine serum albumin (BSA) or its chromophore labelled version, fluorescein‐BSA, was used as a model protein and the loading was performed with a simple mixing of pre‐formed nanocapsules and protein. Dynamic light scattering and zeta potential analysis revealed that protein loaded nanocarriers with high positive zeta potential were formed. The protein loaded nanocarriers displayed a loading efficiency of 75% and a very slow protein release. In summary, our results highlight the potential of CHC as a protein nanocarrier, but also indicate that protein‐CHC interactions need to be considered in protein containing CHC formulations where protein release is not the main function. Refereed/Peer-reviewed

The development of new adhesive wound care products intended for an application over a prolonged time requires good water transporting properties of the adhesive for the maintenance of a suitable environment around the wound. The ability of polydimethylsiloxane (PDMS)-based silicone films to transport water has led to its use in skin pressure-sensitive adhesives and it would be advantageous to find ways for controlling or increasing water transport across PDMS films in order to be able to develop improved skin adhesives. In this study we present a way to increase water transport in such films by the addition of hydrophilic excipients. Three hydrophilic additives, highly water-soluble sucrose and the two superabsorbent polymers (SAP) Carbopol® and Pemulen™, were investigated. The effect of the excipients was characterized by water transport studies, swelling tests, scanning electron microscopy imaging and confocal microscopy. The cross-linked polymers, primarily Pemulen™, were efficient water transport enhancers, whereas sucrose did not show any effect. The effect of the additives seemed to correlate with their water binding capacity. For SAPs the formation of a percolating structure by swollen polymer was also suggested, which enhances water penetration by the higher volume fraction of areas with a higher diffusion constant (swollen SAP), leading to a faster transport through the entire film. Refereed/Peer-reviewed

We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595 nm) can be identified and corrected by recording absorption spectra in the region of 350–850 mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595 nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595 nm by measuring the sample absorbance at 850 nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595 nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.

Adhesion is an important property for the functionality of many medical devices. One reason for the development of adhesive forces is dehydration caused by mass transport of water. Osmotic pressure is one main driving force for mass transport and the correlation between osmotic pressure and adhesive force has not been studied yet, which was the aim of the present study. A model system was used where a Carbopol tablet was lowered onto a 1% (w/w) agarose gel. The force required to detach the tablet (adhesive force) and the weight gain of the tablet (as a measure of transported water) were determined. Sodium chloride and mannitol were added to the agarose gel to decrease the osmotic pressure difference between the agarose gel and the partially hydrated Carbopol tablet. This resulted in a decrease of both mass transport and adhesive force. In addition, experiments with restricted water transport within the agarose gel were performed by preparing gels with different agarose concentrations. An increase of the agarose concentration resulted in decreased water transport and higher adhesive forces. Hence, the results confirmed our hypothesis that osmotic-driven mass transport and restricted mass transport of water correlate very well with the adhesive force.