Mucin-Like Glycoproteins Modulate Interfacial Properties of a Mimetic Ocular Epithelial Surface.

Dry eye disease (DED) has high personal and societal costs, but its pathology remains elusive due to intertwined biophysical and biochemical processes at the ocular surface. Specifically, mucin deficiency is reported in a subset of DED patients, but its effects on ocular interfacial properties remain unclear. Herein a novel in vitro mucin-deficient mimetic ocular surface (Mu-DeMOS) with a controllable amount of membrane-tethered mucin molecules is developed to represent the diseased ocular surfaces. Contact angle goniometry on mimetic ocular surfaces reveals that high surface roughness, but not the presence of hydrophilic mucin molecules, delivers constant hydration over native ocular surface epithelia. Live-cell rheometry confirms that the presence of mucin-like glycoproteins on ocular epithelial cells reduces shear adhesive strength at cellular interfaces. Together, optimal surface roughness and surface chemistry facilitate sustainable lubrication for healthy ocular surfaces, while an imbalance between them contributes to lubrication-related dysfunction at diseased ocular epithelial surfaces. Furthermore, the restoration of low adhesive strength at Mu-DeMOS interfaces through a mucin-like glycoprotein, recombinant human lubricin, suggests that increased frictional damage at mucin-deficient cellular surfaces may be reversible. More broadly, these results demonstrate that Mu-DeMOS is a promising platform for drug screening assays and fundamental studies on ocular physiology.

Sotrastaurin and cyclosporine drug interaction study in healthy subjects.

INTRODUCTION: Sotrastaurin is an immunosuppressant that inhibits protein kinase C and blocks T-lymphocyte activation. The authors determined the effect of combining sotrastaurin with the calcineurin inhibitor cyclosporine on the pharmacokinetics and biomarker responses to both drugs. METHODS: This was a randomized, 4-period, crossover study in 20 healthy subjects who received single oral doses of (1) sotrastaurin 100 mg, (2) cyclosporine 400 mg, (3) 100 mg sotrastaurin with 100 mg cyclosporine and (4) 100 mg sotrastaurin with 400 mg cyclosporine. Blood samples were collected to measure drug levels and biomarkers of T-lymphocyte activation (interleukin-2 and tumor necrosis factor producing T-cells and interleukin-2 messenger RNA levels) and of T-lymphocyte proliferation (thymidine uptake). RESULTS: Sotrastaurin did not alter cyclosporine AUC; however, low-dose and high-dose cyclosporine increased sotrastaurin AUC by 1.2-fold [90% confidence interval, 1.1-1.4] and 1.8-fold [1.6-2.1], respectively. Adding high-dose cyclosporine to a low-therapeutic dose of sotrastaurin significantly enhanced the inhibition of cytokine production by 31% [95% confidence interval, 25-36%], of interleukin-2 messenger RNA levels by 13% [7-19%], and of thymidine uptake by 37% [32-42%] compared with sotrastaurin alone. Addition of low-dose cyclosporine elicited slightly lower enhancements in inhibition by 21% [14-28%], 6% [-4-16%], and 26% [21-30%], respectively, compared with sotrastaurin alone. CONCLUSIONS: Sotrastaurin did not alter the pharmacokinetics of cyclosporine, but cyclosporine increased sotrastaurin AUC up to 1.8-fold. The combined drugs elicited a significantly greater inhibition of T-cell activation and proliferation than sotrastaurin alone.

Understanding the adsorption and potential tear film stability properties of recombinant human lubricin and bovine submaxillary mucins in an in vitro tear film model.

The wetting and adsorption properties for two glycoproteins, recombinant human lubricin and bovine submaxillary mucins (BSM) were evaluated on hydrophilic and hydrophobic glass dome surfaces in a simplified in vitro tear film model. We show that both recombinant human lubricin (rh-lubricin) and BSM solutions render surfaces hydrophilic and when the fluid films reach 500 nm or less, the fluids resist evaporation-driven breakup through a volumetric flux across the surface, which we believe is due to evaporation-driven solutocapillary flows. rh-Lubricin was able to maintain a wet film without spontaneous breakup for longer periods of time than BSM at lower concentrations, which we attribute to differences in adsorption properties, measured by QCM-D, that result from surface charge and structural differences (confirmed by zeta potential, DLS, and SAXS measurements).

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry.

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6-9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Sotrastaurin and tacrolimus coadministration: effects on pharmacokinetics and biomarker responses.

Sotrastaurin is an immunosuppressant that inhibits protein kinase C. In the prevention of acute rejection in organ transplantation, sotrastaurin might be combined with tacrolimus. A drug interaction study was performed in 18 healthy subjects who received single oral doses of sotrastaurin 400 mg, tacrolimus 7 mg, and the drug combination. Drug blood levels and lymphocyte activation and proliferation were measured. Tacrolimus did not alter the pharmacokinetics of sotrastaurin; however, sotrastaurin increased tacrolimus area under the concentration-time curve by 2.0-fold (90% confidence interval, 1.8-2.1). Production of interleukin-2 and tumor necrosis factor by T cells activated via calcium-independent pathways was inhibited by 75% ± 22% from baseline by sotrastaurin. Interleukin-2 messenger RNA levels were decreased by 90% ± 9% from baseline by sotrastaurin. Addition of tacrolimus to sotrastaurin had minimal or no effect on these biomarkers, consistent with tacrolimus' mechanism of action. Lymphocyte proliferation induced via calcium-dependent pathways was decreased from baseline by 82% ± 9% by sotrastaurin, 76% ± 11% by tacrolimus, and 96% ± 2% for the drug combination. How sotrastaurin and tacrolimus could be partnered in an immunosuppressive regimen will need to be established in the context of controlled clinical trials in organ transplant patients, taking into account the pharmacokinetic interaction on tacrolimus and the potentially enhanced immunosuppressive activity of this drug combination.

Contact allergens and irritants show discrete differences in the activation of human monocyte-derived dendritic cells: consequences for in vitro detection of contact allergens.

In recent years test systems have been described that may be applied routinely to discriminate between contact allergens and irritants in vitro. Using human monocyte-derived dendritic cells (MoDC), this study was designed to refine the settings of a potential routine screening protocol for contact allergens and to investigate the so far poorly defined concentration dependency of contact allergen-specific effects. MoDC were generated by 6 days of culture in the presence of IL-4 and GM-CSF and were then cultured for 24 or 48 h in medium with lipopolysaccharide (LPS), contact allergens [picrylsulphonic acid (TNBS), 1-chloro-2,4-dinitrobenzene (DNCB)] or irritants [sodium dodecyl sulphate (SDS), benzalkonium chloride (BAC)] that were free of detectable endotoxin contamination. The induction of CD86 and HLA-DR expression was quantified by flow cytometry as markers for MoDC activation. LPS activation upregulated CD86 about 20-fold and HLA-DR expression about 4-fold. Compared to LPS, contact allergens had weaker effects. TNBS and DNCB induced activation marker upregulation starting slightly below the cytotoxic concentration and increasing in a dose-dependent manner. However, at partially cytotoxic concentrations, irritants also induced CD86 and HLA-DR expression, as confirmed by flow cytometry and quantitative RT-PCR. Both SDS and BAC induced activation marker expression on surviving MoDC, when more than 50% of the MoDC population had been killed by the treatment. Consequently, routine testing of unknown substances would need to quantify activation marker expression as well as cytotoxicity in parallel. In the concentration range around the lowest cytotoxic concentration, the assay may be able to discriminate between contact allergens and irritants.