Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Simple, sensitive and specific quantification of diamine oxidase activity in complex matrices using newly discovered fluorophores derived from natural substrates.

OBJECTIVE: To measure diamine oxidase (DAO) activity with high sensitivity in complex matrices like plasma or tissue extracts radioactive putrescine or horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) coupling must be used. The use of radioactive material should be avoided and HRP/H2O2 coupling is compromised by antioxidants. METHODS AND RESULTS: Condensation of ortho-aminobenzaldehyde (oABA) with delta-1-pyrroline and delta-1-piperideine, the autocyclization products of the DAO-oxidized natural substrates putrescine and cadaverine, generates new quinazoline fluorophores with absorption and excitation maxima of 430 and 460 nm, respectively, and peak emission at 620 nm. Fluorescent-based detection limits are 20-40 times lower compared to absorption measurements. This assay can be used to measure DAO activity in human plasma after spiking recombinant human (rh)DAO, in rat plasma after intravenous rhDAO administration, in pregnancy plasma and in tissue extracts of DAO wild-type and knock-out mice. Using rat plasma the correlation between rhDAO activity and ELISA data is 99%. Human and rat plasma without DAO spiking and tissue extracts from DAO knock-out mice showed stable and low fluorescence in the presence of high substrate concentrations. CONCLUSIONS: Incubation of DAO with the natural substrates putrescine and cadaverine and oABA generates novel fluorophores increasing the detection limit compared to absorption measurements at least tenfold. This simple, sensitive and specific assay allows the non-radioactive quantification of DAO activity in complex matrices like plasma and tissue extracts without interference by antioxidants.

The p-rpS6-zone delineates wounding responses and the healing process.

The spatial boundaries of tissue response to wounding are unknown. Here, we show that in mammals, the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury, forming a zone of activation surrounding the region of the initial insult. This p-rpS6-zone forms within minutes after wounding and is present until healing is complete. The zone is a robust marker of healing as it encapsulates features of the healing process, including proliferation, growth, cellular senescence, and angiogenesis. A mouse model that is unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in impaired healing, identifying p-rpS6 as a modulator but not a driver of healing. Finally, the p-rpS6-zone accurately reports on the status of dermal vasculature and the effectiveness of healing, visually dividing an otherwise homogeneous tissue into regions with distinct properties.

Bio-Distribution and Pharmacokinetics of Topically Administered γ-Cyclodextrin Based Eye Drops in Rabbits.

The purpose of this study was to evaluate the ocular pharmacokinetics, bio-distribution and local tolerability of γ-cyclodextrin (γCD) based irbesartan 1.5% eye drops and candesartan 0.15% eye drops after single and multiple topical administration in rabbit eyes. In this randomized, controlled study, a total number of 59 New Zealand White albino rabbits were consecutively assigned to two study groups. Group 1 (n = 31) received irbesartan 1.5% and group 2 (n = 28) candesartan 0.15% eye drops. In both groups, single dose and multiple administration pharmacokinetic studies were performed. Rabbits were euthanized at five predefined time points after single-dose administration, whereas multiple-dose animals were dosed for 5 days twice-daily and then euthanized 1 h after the last dose administration. Drug concentration was measured by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the retinal tissue, vitreous humor, aqueous humor, corneal tissue and in venous blood samples. Pharmacokinetic parameters including maximal drug concentration (Cmax), time of maximal drug concentration (Tmax), half-life and AUC were calculated. To assess local tolerability, six additional rabbits received 1.5% irbesartan eye drops twice daily in one eye for 28 days. Tolerability was assessed using a modified Draize test and corneal sensibility by Cochet Bonnet esthesiometry. Both γCD based eye drops were rapidly absorbed and distributed in the anterior and posterior ocular tissues. Within 0.5 h after single administration, the Cmax of irbesartan and candesartan in retinal tissue was 251 ± 142 ng/g and 63 ± 39 ng/g, respectively. In the vitreous humor, a Cmax of 14 ± 16 ng/g for irbesartan was reached 0.5 h after instillation while Cmax was below 2 ng/g for candesartan. For multiple dosing, the observed Cmean in retinal tissue was 338 ± 124 ng/g for irbesartan and 36 ± 10 ng/g for candesartan, whereas mean vitreous humor concentrations were 13 ± 5 ng/g and <2 ng/g, respectively. The highest plasma concentrations of both irbesartan (Cmax 5.64 ± 4.08 ng/mL) and candesartan (Cmax 4.32 ± 1.04 ng/mL) were reached 0.5 h (Tmax) after single administration. Local tolerability was favorable with no remarkable differences between the treated and the control eyes. These results indicate that irbesartan and candesartan in γCD based nanoparticle eye drops can be delivered to the retinal tissue of the rabbit's eye in pharmacologically relevant concentrations. Moreover, safety and tolerability profiles appear to be favorable in the rabbit animal model.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Cutaneous optical coherence tomography for longitudinal volumetric assessment of intradermal volumes in a mouse model.

Clinical evaluation of skin lesions requires precise and reproducible technologies for their qualitative and quantitative assessment. In this study, we investigate the applicability of a custom-built dermatologic OCT system for longitudinal assessment of intradermal volumes in a mouse model. The OCT, based on an akinetic swept laser working at 1310 nm was employed for visualization and quantification of intradermal deposits of three different hyaluronic acid-based hydrogel formulations - one commercial and two test substances. Hydrogels were applied in 22 BALB/c mice, and measurements were performed over a six-month time period. All hydrogels increased in volume within the first weeks and degraded steadily thereafter. The half-lifes of the test hydrogels (27.2 ± 13.6 weeks for Hydrogel 1, 31.5 ± 17.2 weeks for Hydrogel 2) were higher in comparison to the commercially available HA hydrogel (21.4 ± 12.0 weeks), although differences were not significant. The sphericity parameter was used for evaluation of the deposit geometry. While on the injection day the sphericities were similar (~0.75 ± 0.04), at later time points significant differences between the different test substances were found (T24: PRV 0.59 ± 0.09, Hydrogel 1 0.70 ± 0.11, Hydrogel 2 0.78 ± 0.07; p ≤ 0.012 for all pairs). This study shows the applicability of OCT imaging for quantitative assessment of the volumetric behavior of intradermal deposits in vivo.

Comparison of optical coherence tomography and high frequency ultrasound imaging in mice for the assessment of skin morphology and intradermal volumes.

Optical coherence tomography (OCT) and high-frequency ultrasound (HFUS), two established imaging modalities in the field of dermatology, were evaluated and compared regarding their applicability for visualization of skin tissue morphology and quantification of murine intradermal structures. The accuracy and reproducibility of both methods were assessed ex vivo and in vivo using a standardized model for intradermal volumes based on injected soft tissue fillers. OCT revealed greater detail in skin morphology, allowing for detection of single layers due to the superior resolution. Volumetric data measured by OCT (7.9 ± 0.3 µl) and HFUS (7.7 ± 0.5 µl) were in good agreement and revealed a high accuracy when compared to the injected volume of 7.98 ± 0.8 µl. In vivo, OCT provided a higher precision (relative SD: 26% OCT vs. 42% HFUS) for the quantification of intradermal structures, whereas HFUS offered increased penetration depth enabling the visualization of deeper structures. A combination of both imaging technologies might be valuable for tumor assessments or other dermal pathologies in clinical settings.