Pharmacological characterisation of small molecule RGD-mimetics targeting αvβ6-mediated activation of TGFβ in idiopathic pulmonary fibrosis

Previously held under moratorium in Chemistry department (GSK) from 11th January 2016 until 18 June 2021.Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include idiopathic pulmonary fibrosis (IPF). The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of IPF. Therefore, the αvβ6 integrin was identified as an attractive therapeutic target for this debilitating disease and a drug discovery programme to identify inhaled small molecule αvβ6 selective arginylglycinyl- aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the small molecule αvβ6 RGD-mimetics generated in a range of test systems. RGD ligands including peptides, antibodies and small molecule RGD-mimetics were characterised in vitro using methods that included radioligand binding, cell adhesion, flow cytometry, coculture TGFβ bioassays, confocal microscopy and immunohistochemistry. This included the use of soluble recombinant integrin protein, stable and primary cells expressing integrins recombinantly or endogenously and human lung tissue from normal and IPF donors. Compound 3 generated from an internal medicinal chemistry programme was shown to bind to the αvβ6 with high affinity (pM) and displayed fast association with the integrin followed by slow dissociation kinetics in all systems investigated. Using [3H]compound 3 the quantification of the upregulation of αvβ6 from normal to IPF human lung tissue has been determined for the first time with data suggesting αvβ6 exists in a highly activated state within diseased tissue. In human lung primary epithelial cells co-cultured with a TGFβ reporter cell system, compound 3 was shown to inhibit with high potency the activation of TGFβ with a prolonged duration of action. In the same primary lung epithelial cells compound 3 caused a rapid internalisation of αvβ6 (minutes) followed by a slow return of the integrin to the cell surface (hours). It was shown that αvβ6 is degraded in lysosomes post-internalisation by compound 3 that would suggest the slow return of integrin to the surface and sustained duration of action is a consequence of new αvβ6 synthesis. From the chemical series of small molecule RGD-mimetic compounds identified, compound 3 has been shown to display the desirable pharmacological characteristics required for targeting a prolonged inhibition of TGFβ activation in the IPF lung via blockade of the αvβ6 integrin.Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include idiopathic pulmonary fibrosis (IPF). The alpha-v beta-6 (αvβ6) integrin has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of IPF. Therefore, the αvβ6 integrin was identified as an attractive therapeutic target for this debilitating disease and a drug discovery programme to identify inhaled small molecule αvβ6 selective arginylglycinyl- aspartic acid (RGD)-mimetics was initiated within GlaxoSmithKline. The primary aim of this study was to pharmacologically characterise the small molecule αvβ6 RGD-mimetics generated in a range of test systems. RGD ligands including peptides, antibodies and small molecule RGD-mimetics were characterised in vitro using methods that included radioligand binding, cell adhesion, flow cytometry, coculture TGFβ bioassays, confocal microscopy and immunohistochemistry. This included the use of soluble recombinant integrin protein, stable and primary cells expressing integrins recombinantly or endogenously and human lung tissue from normal and IPF donors. Compound 3 generated from an internal medicinal chemistry programme was shown to bind to the αvβ6 with high affinity (pM) and displayed fast association with the integrin followed by slow dissociation kinetics in all systems investigated. Using [3H]compound 3 the quantification of the upregulation of αvβ6 from normal to IPF human lung tissue has been determined for the first time with data suggesting αvβ6 exists in a highly activated state within diseased tissue. In human lung primary epithelial cells co-cultured with a TGFβ reporter cell system, compound 3 was shown to inhibit with high potency the activation of TGFβ with a prolonged duration of action. In the same primary lung epithelial cells compound 3 caused a rapid internalisation of αvβ6 (minutes) followed by a slow return of the integrin to the cell surface (hours). It was shown that αvβ6 is degraded in lysosomes post-internalisation by compound 3 that would suggest the slow return of integrin to the surface and sustained duration of action is a consequence of new αvβ6 synthesis. From the chemical series of small molecule RGD-mimetic compounds identified, compound 3 has been shown to display the desirable pharmacological characteristics required for targeting a prolonged inhibition of TGFβ activation in the IPF lung via blockade of the αvβ6 integrin

Relationship of Weed Control and Soil pH to No-Tillage Corn Yields

Atrazine and simazine are used for selective control of a broad spectrum of weeds in corn. Over 80% of the U.S. corn production is treated with one or the other of these two s-triazine herbicides. In Kentucky they are used annually on over 800,000 acres of corn, including over 200,000 acres of no-tillage corn. When added to the soil these compounds are ultimately degraded to non-phytotoxic compounds. The rate of degradation is dependent upon the physical, chemical, and biological properties of the soil. Although atrazine and simazine are chemically similar, simazine is considered to degrade slightly slower than atrazine after application to the soil and as a result will remain in the soil for a longer period of time

Post-Operative Medium- and Long-Term Endocrine Outcomes in Patients with Non-Functioning Pituitary Adenomas—Machine Learning Analysis

Post-operative endocrine outcomes in patients with non-functioning pituitary adenoma (NFPA) are variable. The aim of this study was to use machine learning (ML) models to better predict medium- and long-term post-operative hypopituitarism in patients with NFPAs. We included data from 383 patients who underwent surgery with or without radiotherapy for NFPAs, with a follow-up period between 6 months and 15 years. ML models, including k-nearest neighbour (KNN), support vector machine (SVM), and decision tree models, showed a superior ability to predict panhypopituitarism compared with non-parametric statistical modelling (mean accuracy: 0.89; mean AUC-ROC: 0.79), with SVM achieving the highest performance (mean accuracy: 0.94; mean AUC-ROC: 0.88). Pre-operative endocrine function was the strongest feature for predicting panhypopituitarism within 1 year post-operatively, while endocrine outcomes at 1 year post-operatively supported strong predictions of panhypopituitarism at 5 and 10 years post-operatively. Other features found to contribute to panhypopituitarism prediction were age, volume of tumour, and the use of radiotherapy. In conclusion, our study demonstrates that ML models show potential in predicting post-operative panhypopituitarism in the medium and long term in patients with NFPM. Future work will include incorporating additional, more granular data, including imaging and operative video data, across multiple centres

Pharmacological characterization of the αvβ6 integrin binding and internalization kinetics of the foot-and-mouth disease virus derived peptide A20FMDV2

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alphav beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand- binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (KD:0.22nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and LAP1 was shown to be fast (t1/2:1.5and 3.1 min, respectively), concentration-dependent (EC50:values 1.1 and 3.6nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand-binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement

Switchable mirrors based on nickel-magnesium films

A new type of electrochromic mirror electrode based on reversible uptake of hydrogen in nickel magnesium alloy films is reported. Thin,magnesium-rich Ni-Mg films prepared on glass substrates by cosputtering from Ni and Mg targets are mirror-like in appearance and have low visible transmittance. Upon exposure to hydrogen gas or on reduction in alkaline electrolyte, the films take up hydrogen and become transparent. When hydrogen is removed, the mirror properties are recovered. The transition is believed to result from reversible formation of Mg2NiH4 and MgH2. A thin overlayer of palladium was found to enhance the kinetics of hydrogen insertion and extraction,and to protect the metal surface against oxidation

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

Background: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: ‘‘rigidity dependent’ ’ (those which show an increase in cell growth as extracellular rigidity is increased), and ‘‘rigidity independent’’ (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance: These observations suggest that the mechanical properties of the matrix environment play

Haemophilus influenzae Type b Vaccine Failure in Children Is Associated with Inadequate Production of High-Quality Antibody

Background. Despite the excellent immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines, breakthrough cases of Hib disease still affect a small proportion of vaccinated children in the United Kingdom. We performed a retrospective study to compare the avidity of antibody directed against the Hib polysaccharide capsule (PRP) in children who experienced Hib vaccine failure in the United Kingdom among 3 historical cohorts and with age-matched healthy control subjects. Methods. Serum samples from vaccinated children with invasive Hib disease were collected beginning in 1992 as part of enhanced surveillance for Hib disease following vaccine introduction. A total of 251 children who experienced Hib vaccine failure were identified from 3 historical cohorts (1992-1995, 1996-1999, and 2000-2003). The anti-PRP antibody concentration and avidity from healthy age-matched control subjects was obtained for the 3 contemporary time points (1995, 1999, and 2002). Serum anti-PRP antibody concentration was measured in each of the samples using a standard Hib ELISA, and antibody avidity was determined using thiocyanate elution. Results. Within the first 60 days after disease onset, there was no change in the anti-PRP antibody avidity, and there was no statistically significant difference in the geometric mean Hib antibody avidity over the 3 study periods. However, the children who experienced Hib vaccine failure had significantly lower Hib antibody avidity than did healthy control subjects, despite a marked antibody response following infection. Conclusions. Children who experience Hib disease despite vaccination appear to have a defect in immunological priming, leading to a qualitative difference in Hib-specific memory B cells. Low anti-PRP antibody avidity decreases the functional activity of anti-PRP antibody in the sera of these children experiencing vaccine failure, leading to disease susceptibilit

Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1–30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI