Topical Application of an Irreversible Small Molecule Inhibitor of Lysyl Oxidases Ameliorates Skin Scarring and Fibrosis

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases

Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms

The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10–40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response.Published versio

Pan-Lysyl Oxidase Inhibitor PXS-5505 Ameliorates Multiple-Organ Fibrosis by Inhibiting Collagen Crosslinks in Rodent Models of Systemic Sclerosis

Systemic sclerosis (SSc) is characterised by progressive multiple organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for myelofibrosis treatment was evaluated using in vivo rodent models resembling the fibrotic conditions in SSc. Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression were elevated in the skin and lung of SSc patients. The oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced pulmonary fibrosis toward normal levels, mediated by its ability to normalise collagen/elastin crosslink formation. PXS-5505 also reduced fibrotic extent in models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney, and the CCl4-induced fibrotic liver. PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc

Biofilm-associated persister bacteria in <i>P. aeruginosa</i>.

<p>(A) Young <i>P. aeruginosa</i> biofilms pre-grown for 5 h in the absence of antibiotic before being treated with tobramycin at 0-160 mg/L for 1-3 h. Biofilm viability was analysed by the drop plate method and CFU counts. Error bars indicate standard error of the geometric mean (SEM, n = 3). (B) Same as in (A) but established <i>P. aeruginosa</i> biofilms were pre-grown for 20 h in the absence of antibiotic before being treated with tobramycin. Error bars indicate standard deviation (SD, n = 4).</p

Mannitol alone does not induce biofilm dispersal or decrease in biofilm CFU counts.

<p>(A) Biofilms were pre-grown for 5 h in microtitre plates in the absence of any treatment. Then individual treatments with mannitol, glucose, NaCl or tobramycin as a positive control were added to the wells and the plates incubated for further 2 h before analysing biofilm viability by the drop plate method and CFU counts. Error bars indicate SD (n = 4). (B) Microscopic images of LIVE/DEAD stained <i>P. aeruginosa</i> biofilms grown at the bottom of 24-well plates for 6 h or 24 h in the presence or absence of 40 mM mannitol. Live cells appear green, dead cells appear red. Note that the DEAD stain is also known to bind extracellular DNA in the biofilm matrix [51]. Bar, 20 µm. (C) <i>P. aeruginosa</i> biofilms were cultivated in continuous flow microfermenters with glucose at 2 mM. After 1 day of growth, the medium inlet was supplemented (arrow) with glucose at 20 mM (opened circles), 100 mM (opened squares), or mannitol at 20 mM (filled upright triangles) or 100 mM (filled inverted triangles) and the release of dispersal cell was monitored by measuring the OD₆₀₀ of the effluent runoff. </p

Mannitol increases the efficacy of tobramycin against clinically relevant strain <i>P. aeruginosa</i> FRD1, but not tobramycin resistant <i>P. aeruginosa</i> 18A, which was isolated from a CF patient.

<p>(A) Biofilms were grown in multiwell plates for 5 h (FRD1) or 24 h (18A) at which time tobramycin was added to the cultures at various concentrations. The plates were incubated for a further 2 h before enumerating CFU. (B) Biofilms were grown for 5 h (FRD1) or 24 h (18A) in the absence of antibiotic, before being treated with or without tobramycin at 80 mg/L (FRD1) or 400 mg/L (18A) for 1 h to select for persisters. Then mannitol was directly added to the wells at 0-40 mM, and the plates were incubated for a further 2 h before analysing CFU. Error bars indicate SD (n = 4). Asterisks indicate statistically significant difference of combination treatments versus tobramycin only (**, <i>P</i> < 0.01; ***, <i>P</i> < 0.001).</p

Exposure to mannitol reverses the persister phenotype in <i>P. aeruginosa</i> biofilms.

<p>(A) Young, 5 h old biofilms grown in multiwell plates were first treated with or without 80 mg/L tobramycin for 1 h to select for persister cells. Then mannitol was added to the culture and the plates were incubated for a further 1-2 h, before enumerating CFU. Glucose and NaCl were used in place of mannitol to check for substrate or osmotic effects. (B) Established, starving biofilms grown for 20 h in the absence of treatment were exposed or not to tobramycin for 1 h. Mannitol, glucose or NaCl were then added the cultures for a further 1-3 h, before analysing CFU. Error bars indicate SEM (n is indicated in parentheses for each set of samples). Asterisks indicate statistically significant difference of combination treatments versus tobramycin only (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001).</p

Reducing safety-related drug attrition: The use of in vitro pharmacological profiling

In vitro pharmacological profiling is increasingly being used earlier in the drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the development of candidate drugs or even lead to market withdrawal if discovered after a drug is approved. Here, for the first time, the rationale, strategies and methodologies for in vitro pharmacological profiling at four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis and Pfizer) are presented and illustrated with examples of their impact on the drug discovery process. We hope that this will enable other companies and academic institutions to benefit from this knowledge and consider joining us in our collaborative knowledge sharing