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 minutes. 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.
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 (H 2 O 2 ) coupling must be used. The use of radioactive material should be avoided and HRP/H 2 O 2 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. Electronic supplementary material The online version of this article (10.1007/s00011-020-01359-5) contains supplementary material, which is available to authorized users.
<strong>Background:</strong> 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. <strong>Results:</strong> Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After replacement of positively charged amino acids of the heparin-binding motif with polar serine or threonine residues 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. <strong>Conclusions: </strong>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. <strong>Funding: </strong>Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); ADD funding Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).
Intravenous (IV) administration in mice is predominantly performed via the lateral tail veins. The technique requires adequate training before it can be used safely and routinely. A novel anaesthesia induction chamber has been developed to simplify the treatment and to facilitate IV injection in mice, particularly for untrained personnel. We have assessed the benefits of the chamber in refining IV injection in isoflurane-anaesthetized mice in direct comparison with the common restrainer method on conscious animals. The body weight, nesting behaviour and concentrations of faecal corticosterone metabolites were taken as indicative of distress induced by the various procedures. The results suggest that both methods of tail-vein injection induce similar levels of momentary stress in the animals, revealed by a short-term increase in the levels of stress hormone metabolites in faeces. A temporary reduction of body weight was observed after IV injection under isoflurane anaesthesia but not for conscious mice injected in the common restrainer. We conclude that the severity of tail-vein injection in mice is 'mild' for both methods. There was no evidence that refining the procedure by using isoflurane anaesthesia in the induction chamber was associated with any benefit.
4659 Baxter has developed a recombinant FVIIa (rFVIIa) product for the treatment of patients with hemophilia A and hemophilia B with inhibitors. The aim of the presented preclinical studies was to evaluate the efficacy of Baxter's rFVIIa. Four animal models that are considered to relevantly reflect the conditions in different patient populations were used: hemophilia A (FVIII ko) mice, hemophilia B (FIX ko) mice, factor VIII (FVIII)-inhibited rabbits and warfarin-pretreated rats. In all studies, Baxter's rFVIIa was tested at different doses to obtain dose-effect curves. A commercially available rFVIIa served as the active control item, buffer served as the negative control item. All test and control items were administered intravenously. Mice received prophylactic treatment 5 minutes prior to start of the experiment, rabbits and rats received therapeutic treatment after the injuries were inflicted. Twenty FVIII ko mice (B6;129S4-F8tm1Kaz; 10m/10f) and 20 FIX ko mice (B6;129P2-F9tm1Dws; 10m/10f) per group were used in a tail-tip bleeding model. Baxter's rFVIIa was tested at doses of 0.6, 1.2 and 2.7 mg/kg, the active control item was tested at a dose of 2.7 mg/kg. Ten FVIII ko mice (B6;129S4-F8tm1Kaz; 5m/5f) per group were used to assess the influence of rFVIIa on the thrombelastogram of hemophilic mice. Both items were tested at doses of 0.1, 0.6 and 1.2 mg/kg. Six rabbits (New Zealand White; 6m) per group were used in a nail-cut model. The animals were pretreated with a goat polyclonal FVIII-antibody (34,000 BU/kg i.v., 45 minutes prior to start of the experiment), leading to deficiency of endogenous FVIII. Baxter's new rFVIIa was tested at doses of 0.1, 1.0, 2.0, 2.5 and 3.0 mg/kg, the active control item at 2.0 mg/kg. Six rats (Sprague Dawley; 6m) per group were used in a tail-tip bleeding model. The animals were pretreated with warfarin (11 mg p.o., 1 day prior to start of the experiment), leading to a deficiency of vitamin K-dependent coagulation factors II, VII, IX and X, protein C and protein S. Baxter's new rFVIIa was tested at 0.25, 1.0 and 2.0 mg/kg, the active control item at 2.0 mg/kg. A dose-dependent hemostatic effect of Baxter's new rFVIIa could be shown in all primary pharmacodynamic studies performed. Furthermore, statistical evaluation of the efficacy of Baxter's new rFVIIa did not reveal any statistically significant differences from the commercially available rFVIIa product after treatment at the same doses. In summary, the results of our studies show that Baxter's new rFVIIa is prophylactically and therapeutically effective in animal models of efficacy closely reflecting the conditions in hemophiliacs with inhibitors and patients suffering from FVII-deficiency. Disclosures: Schiviz: Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Resch:Baxter Innovations GmbH: Employment. Leidenmuehler:Baxter Innovations GmbH: Employment. Bischetsrieder:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.