All experiments and procedures were performed within the Division of Biomedical Research, Medical University of Vienna, according to the animal protocol GZ 2021–0.209.778, approved by the Austrian Ministry of Education, Science, and Research.

Animals, housing, and acclimatization

Thirty-eight female Dunkin Hartley guinea pigs (Charles River Laboratories, Sulzfeld, Germany) weighing 465–625 g were housed for at least 14 days prior to the experiment in groups of four in cages containing wooden bedding material. The guinea pigs were fed a commercially available pelleted diet (ssniff Spezialdiäten, Soest, Germany) ad libitum and hay daily. Tap water was available ad libitum. Once a week they received vitamin C (Gatt Koller GmbH, Absam, Austria) through drinking water. The animals were kept in a light/dark cycle of 12:12 h at 20 ± 2 °C and 55 ± 2% humidity.

Surgical procedure

The guinea pigs were anesthetized subcutaneously (0.1 mg/kg medetomidine [Domitor, Provet, Switzerland], 1 mg/kg midazolam [Midazolam Accord, Accord Healthcare, Austria], 0.03 mg/kg fentanyl [Fentanyl Primal, Primal Critical Care, Netherlands] and 10 mg/kg ketamine [Ketanest, Pfizer, Austria]) by the animal keeper, who received the mixture from the veterinarian. The animals were returned to the cage until the righting reflex disappeared and then transported to the operating room. In preparation for surgery, the ventral side of the neck was shaved and disinfected, and a local anesthetic (lidocaine 1%, 4 mg/kg; Xylanest purum, Gebro Pharma, Austria) was injected subcutaneously. Animals were placed on a heating pad (37 °C) to prevent hypothermia and a rectal probe was inserted to monitor body temperature. Guinea pigs received 0.7 L/min of oxygen through a face mask and a pulse oximeter was attached to the hind limb to measure blood oxygen saturation. After the disappearance of the foot withdrawal reflex, a double-lumen catheter (Nutriline Twinflow 2Fr, Vygon, Germany) was inserted into the right or left jugular vein for intravenous administration of anesthesia and experimental compounds. To maintain anesthesia, guinea pigs were administered a mixture of etomidate (0.2 mg/kg/min; Etomidate-Lipuro, 2 mg/ml, Braun, Germany) and fentanyl (0.03 mg/kg/h; diluted to 20 µg/mL) via the jugular vein catheter as a continuous drip infusion. An additional catheter (Nabelkatheter 2.5 Fr, Vygon, Germany) was placed in the right or left carotid artery for invasive blood and HR measurements and for blood sampling (Supplementary Fig. 1). HR, MAP, and body core temperature (BT) were measured every minute or every three minutes and are presented after normalization to baseline.

Citrate blood (0.3 mL per timepoint, 3.8% sodium citrate) was collected before intravenous injection of test substances (minute 0) and thereafter every 10 min. For histamine measurements, an additional citrate blood sample (0.2 mL per timepoint, 3.8% sodium citrate) was collected every 10 min with the highly potent and specific DAO-inhibitor diminazene aceturate (D7770, Sigma-Aldrich, Austria) at a final concentration of 10 µM to prevent histamine degradation. After each blood collection, the arterial catheter was flushed with 0.3 mL alteplase (400 ng/mL diluted in NaCl, Actilyse Cathflo, Boehringer Ingelheim, Austria) to prevent clotting in the catheter lumen.

Arterial blood gases, acid–base status, electrolyte levels, oxygen status, and lactate were measured at minute 0 and at the end of each experiment using a blood gas analyzer (ABL800 Flex, Drott, Austria).

Experimental procedureUnfractionated heparin

After the first blood draw four guinea pigs received an intravenous bolus infusion of unfractionated heparin (500 IE/kg, Gilvasan Pharma, Austria) and blood was sampled after five minutes.

rhDAO_mHBM pharmacokinetics (PK)

Three guinea pigs received rhDAO_mHBM at a concentration of 2 mg/kg and were observed for 90–100 min. The generation and purification of rhDAO_mHBM were recently published [14].

Histamine dose finding

Four guinea pigs received a continuous drip infusion of histamine (#53300, Sigma-Aldrich, Austria). Histamine dosages ranged from 0.2 to 8 µg/kg/min and increased every 10 or 20 min until a measurable clinical response was observed. The observation period was 70–90 min.

DAO prophylaxis

Twelve guinea pigs received either rhDAO_mHBM (2 or 4 mg/kg in 0.05–0.1 mL/100 g of 24 mM HEPES and 73 mM potassium chloride) or buffer (0.05–0.1 mL/100 g of 24 mM HEPES and 73 mM potassium chloride) prophylactically into the artery after the first blood draw (minute 0). After a stabilization time of 10 min, a continuous drip infusion of 8 µg/kg/min histamine was administered for 30 min. At minute 40, the histamine infusion was stopped and the animals were observed until minute 60.

DAO activity measurement

Guinea pig DAO activity from citrate plasma was measured as described [20]. Briefly, citrate plasma was diluted 1:10 in PBS and incubated for 120 min with 1 mM ortho-aminobenzaldehyde (A9628, Sigma-Aldrich, Austria) and either 200 µM cadaverine (#D22606 Sigma-Aldrich, Austria) or PBS. DAO-deaminated cadaverine autocyclizes to delta-1-piperideine and fuses with ortho-aminobenzaldehyde forming a fluorophore, which can be measured at EX440 and EM620 nm. DAO activity was converted into concentration units using an rhDAO standard curve ranging from 0 µg/mL to 5 µg/mL in baseline guinea pig plasma.

Recombinant human DAO ELISA measurements in plasma

The concentrations of rhDAO_mHBM in the plasma of guinea pigs were measured using an ELISA as previously described [21]. Briefly, citrate plasma was diluted 1:80 in 1% human serum albumin in PBS and again 1:5 in low-cross buffer (#100050, Candor Bioscience, Germany). A standard curve using rhDAO_mHBM in baseline guinea pig citrate plasma was used for quantification. A monoclonal mouse anti-human-DAO antibody (a generous gift from Prof. Andrea Quaroni, Cornell University, Ithaca, NY, USA) was coated at 5 µg/mL in 50 mM carbonate buffer (pH 9.6) overnight and the microtiter plates were subsequently blocked using 1% bovine serum albumin in PBS. After washing with 0.1% Tween-20 in PBS (pH 7.4), the samples and standards were incubated at room temperature. A polyclonal rabbit anti-human-DAO serum IgG fraction (in-house generated; not commercially available) and an HRP-labelled donkey anti-rabbit antibody (#SAB3700928, Sigma-Aldrich, Austria) were used for detection. Additionally, 10 µg/mL donkey IgG was used to minimize the background signal (#017–000-003, Jackson ImmunoResearch, United Kingdom). The chemiluminescent substrate SuperSignal™ was used for signal generation (#37069, Thermo Fisher, Austria).

Histamine measurements in the dose-finding phase

During the dose-finding phase histamine concentrations in citrate plasma containing 10 µM diminazene-aceturate were analyzed using the histamine homogeneous time-resolved fluorescence (HTRF) dynamic kit (62HTMDPET, PerkinElmer formerly Cisbio, France) according to the manufacturer’s instructions with a histamine standard curve in baseline guinea pig plasma.

Citrate plasma with 10 µM diminazene aceturate from all other guinea pigs was analyzed using the state-of-the-art enzyme immunoassay (EIA) for histamine quantification. The kit was used according to the instructions provided by the manufacturer (IM2562, Immunotech/Beckman Coulter, Czech Republic) with a histamine standard curve in guinea pig plasma. All measurements were performed in duplicate and all histamine concentrations refer to the histamine base.

Histamine and 1-Methylhistamine measurements by liquid chromatography – tandem mass spectrometry (LC–MS/MS) in plasma and urine

A total of 38 guinea pigs were used for the establishment of surgical methods, substance titration (dose-finding phase), and the main experiment. Determination of analytes in the plasma and urine of guinea pigs was performed by staff members blinded to the treatment allocation. Histamine and 1-Methylhistamine were determined in all baseline urine samples, whereas plasma histamine was analyzed in 12 guinea pigs with seven time points included in the main experiment.

All samples were analyzed using LC–MS/MS in positive mode by multiple reaction monitoring (MRM). More detailed information on the analytical method can be found in the supplementary material.

Statistical analysis

Statistical analyses were performed using GraphPad Prism Version 8.4.0. (GraphPad Software Inc., San Diego, CA, USA). Differences in DAO plasma concentrations were compared using a paired t-test. The area under the curves (AUCs) of individual HR, MAP, and plasma histamine concentrations during the course of an experiment were compared using a two-sided unpaired t-test without Welch’s correction. The increase in urinary histamine from minute 0 to 60 in guinea pigs receiving 8 µg/kg/min histamine was calculated using a paired t-test. Differences in urinary histamine excretion between guinea pigs receiving histamine alone and guinea pigs pretreated with 4 mg/kg rhDAO_mHBM were calculated using a two-sided unpaired t-test without Welch’s correction. Continuous data are presented as mean ± SEM. Statistical significance was defined as p < 0.05.