Safety, tolerability, and pharmacokinetics/-dynamics of the dipeptidyl peptidase 3-inhibiting antibody Procizumab in a first-in-human trial

ABSTRACT

Dipeptidyl peptidase 3 (DPP3), an aminopeptidase that degrades several key cardiovascular mediators, may induce and exacerbate hemodynamic instability during cardiogenic shock. Procizumab (PCZ) is a first-in-class humanized monoclonal antibody that inhibits DPP3 activity. In preclinical shock models, PCZ increased angiotensin metabolite levels, improved cardiovascular function, and increased survival. Here, results are presented for a first-in-human Phase 1 trial (NCT06331884) that evaluated the safety, tolerability, and pharmacokinetics and pharmacodynamics of PCZ. Twenty-four healthy male volunteers were enrolled in a randomized, double-blind, placebo-controlled Phase 1 trial. Participants (n = 6 per group) received placebo or one of three doses of PCZ (3 mg/kg, 6 mg/kg, and 12 mg/kg). Participants were monitored clinically for 24 h after drug administration, as well as at 6 follow-up visits performed during a 28-d period. Adverse events associated with PCZ were predominantly mild, and no serious adverse events were reported. Local tolerability, vital signs, laboratory assessments, and electrocardiograms did not reveal any safety concerns. PCZ exhibited a terminal elimination half-life of 24, 34, and 53 h in the low, intermediate, and high-dose groups, respectively. A volume of distribution of roughly 9.8–10.2 liters indicates that the drug predominantly remains within the circulation. Results of this trial are currently being followed up in the PROCARD Phase 1b/2a (NCT06832722) study, which is evaluating safety and determining the optimal dose of PCZ in cardiogenic shock patients.

Introduction

Shock is a clinical manifestation of circulatory failure, resulting in insufficient oxygen delivery and impaired cellular oxygen utilization, an entity affecting about one third of patients in contemporary intensive care units.¹ Traditionally, shock syndromes are grouped based on the primary causative pathophysiological mechanism: hypovolemic, cardiogenic, obstructive, and distributive.¹ In hypovolemic, cardiogenic, and obstructive shock, initial cardiac output is low, leading to compromised oxygen transport, whereas distributive shock primarily features reduced systemic vascular resistance with compensatory high cardiac output.¹

Although this classification supports initial management, it fails to encompass the complexity and heterogeneity of molecular drivers in shock. Mixed phenotypes, such as cardiogenic-vasodilatory shock, vasodilatory-cardiogenic shock, and primary mixed shock, challenge traditional boundaries, manifesting when overlapping insults trigger combined cardiac dysfunction and vasoplegia.² The prevalence of mixed shock states is rising, with associated systemic inflammation, vasoplegia, and higher mortality, warranting invasive hemodynamic monitoring and prompt identification of underlying causes.^3,4

Despite advances in supportive care (fluid resuscitation, vasoactive therapy, mechanical circulatory support, organ support), the overall prognosis for circulatory failure has not substantially improved over three decades. The development of targeted therapies hinges on delineating biologically meaningful pathways and phenotyping strategies into research, trial design, and clinical practice. This approach aims to address the persistent mortality and morbidity associated with shock syndromes.⁵ Consequently, global research efforts are directed at improving outcomes in shock.^6,7

Dipeptidyl peptidase 3 (DPP3) is an enzyme ubiquitously expressed in human tissues, but only present at low concentrations in the plasma (circulating DPP3; cDPP3) in healthy participants, where it is involved in the degradation of several important regulators of vascular tone.^8,9 High cDPP3 concentrations in patients were found to be strongly associated with impaired outcomes in cardiogenic and septic shock,^9–12 while a decrease of cDPP3 following treatment was associated with a lesser requirement for organ support and lower mortality.^10,11 These clinical associations, combined with the known short half-life and primary cytosolic localization of DPP3,^13,14 imply that high cDPP3 levels in critical illness represent a state of ongoing cell death (necrosis) leading to the uncontrolled release of cytosolic DPP3 into the circulation.⁹ During shock, upregulation of the vasoconstrictive molecule angiotensin II is a physiologic and potentially life-saving response aimed at maintaining adequate tissue perfusion.^15,16 As cDPP3 can effectively degrade Angiotensin II, it may represent a novel factor contributing to hemodynamic instability during shock types with a vasodilatory component.^9,11

Procizumab (PCZ; international nonproprietary name invobenitug) is a DPP3-antagonizing antibody that demonstrated promising results in murine models of sepsis -and cardiogenic shock.^12,17 In a murine isoproterenol-induced model of heart failure, PCZ administration effectively normalized left ventricular function, an effect which persisted up to 14 d.¹² Administration of PCZ in a murine cecal ligation and puncture sepsis model attenuated sepsis-induced cardiac dysfunction and cardiac oxidative stress and improved survival.¹⁷ Extensive preclinical safety and toxicological studies of PCZ did not reveal any safety concerns.^12,17–19

Here, we report the results of a Phase 1, first-in-human study on the safety, tolerability, and pharmacokinetics (PK) and pharmacodynamics (PD) of single escalating intravenous (i.v.) doses of PCZ.

Materials and methods

General

The trial consisted of a first-in-human, Phase 1, randomized, double-blind, placebo-controlled study, evaluating single escalating i.v. doses of PCZ in male healthy participants. The trial was conducted at a single site (Department of Intensive Care Medicine at the Radboud University Medical Center in Nijmegen, the Netherlands) and was carried out in accordance with the Declaration of Helsinki and Good Clinical Practice standards. Permission to carry out this trial was granted by the medical ethics review committee Assen “BEBO foundation” in accordance with European Clinical Trial Regulation (CTR) (EU TRIAL NR.: 2023-507035-37–00). The study was registered at ClinicalTrials.gov (NCT06331884).

Study medication

PCZ is a recombinant, humanized IgG1 monoclonal antibody directed against a conserved, surface exposed loop in proximity to the active enzyme site.¹² In vitro data indicate that PCZ inhibits cDPP3’s function by up to ~85%.^12,20 PCZ and placebo were produced under good manufacturing practice (GMP) conditions and subsequently supplied by the study sponsor (4TEEN4 Pharmaceuticals GmbH, Hennigsdorf, Germany) as a solution for injection in identical sterile single-use vials, containing study medication at 20 mg/mL.

Participants were randomized by an unblinded research team using a predefined block‑randomization list generated at study initiation. Six blocks were used, each containing three active treatments and one placebo, ensuring balanced allocation within each dose group. In each cohort, the first block functioned as a sentinel group, in which subjects were dosed individually with at least 48 h between doses. Since PCZ was not found to represent a high-risk product, no minimally anticipated biological effect level was used for selection of the starting dose. Instead, dose selection for this first-in-human trial was based on preclinical toxicology data, which demonstrated a no observed adverse effect level (NOAEL) of 150 mg/kg (maximum technically feasible dose) and 350 mg/kg for mice and cynomolgus monkeys, respectively. Considering the recommendations provided in the European Medicines Agency Guideline on strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational medicinal products (EMEA/CHMP/SWP/28367/07 Rev. 1), these NOAELs provided a 50- and 116-fold, respectively, safety margin to the investigated starting dose of 3 mg/kg. Subsequent dose levels were determined using an acceptable spacing factor of 2, allowing for safe and controlled dose escalation while adequately covering the expected pharmacological active dose range (i.e., mid dose of 6 mg/kg) and extending beyond to even higher doses (i.e., 12 mg/kg).

Participants

After providing written consent, participants aged 18–35 y, with a body mass index (BMI) between 18 kg/m² and 30 kg/m² were included. Before participation, health status was determined by past medical history, physical examination, 12-lead electrocardiography (ECG), and safety laboratory tests at a screening visit. Exclusion criteria included atopic constitution, use of any medication, significant blood loss, and/or participation in any other clinical trial within 90 d prior to study drug administration. The use of recreational drugs was prohibited from 2 d prior to study drug administration until 28 d afterward. Alcohol was prohibited 24 h before and after study drug administration.

Trial procedures

A graphical summary of all trial procedures is provided in Figure 1. Participants were admitted to the Intensive Care Research Unit early in the morning. A cannula was placed in the lower arm for i.v. infusion of study medication as well as fluids. An intra-arterial cannula was also placed, to facilitate frequent blood withdrawals as well as continuous blood pressure monitoring. During the experimental protocol, heart rate and intra-arterial blood pressure were continuously monitored, while peripheral oxygen saturation (spO2) and temperature were measured every 30 min.

Figure 1.

Graphical overview of the trial procedures. Red blood drops represent sample collection timepoints for determination of safety laboratory parameters as well as cDPP3 and Procizumab (PCZ) concentrations, while orange blood drops represent timepoints at which only cDPP3 and Procizumab (PCZ) concentrations were determined.

In total, 24 participants were assigned to one of three dosing groups. Each dose group consisted of eight participants randomly assigned to receive either PCZ (n = 6) or placebo (n = 2), administered as an i.v. infusion over 2 h. In each incremental dose group, a higher dose of PCZ was assessed (starting with 3 mg/kg, followed by 6 mg/kg and 12 mg/kg).

Participants were admitted to the hospital research facilities up until 24 h after study drug administration, during which frequent blood samples were performed for analyses of PK/PD parameters, as well as various laboratory safety parameters. After hospital discharge, participants returned for further follow-up visits 2, 3, 7, 14, 21, and 28 d after study drug administration. During these follow-up visits, they were checked for adverse events (AEs), their vital signs were measured, and venous blood withdrawals were performed for further determination of PK/PD and laboratory safety parameters.

Safety parameters

Safety and tolerability were the study’s primary endpoints. Frequent safety and tolerability assessments were performed on the experimental day until discharge and during the 28-d follow-up period. Safety parameters included blood pressure, heart rate, and peripheral oxygen saturation.

Local tolerability at the site of i.v. infusion was monitored bi-hourly for up until 10 h after the start of study drug administration. After removal of the catheter, the infusion site was also assessed during each of the six follow-up visits.

Routine hematology, clinical chemistry, and clotting laboratory tests were performed at the Department of Laboratory Medicine of Radboud university medical center. Hematology parameters included hemoglobin, leukocytes, the leukocyte differential, and thrombocytes. Biochemistry parameters included sodium, potassium, creatinine, urea, alkaline phosphatase, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), bilirubin, gamma-glutamyl transferase, creatine-kinase, albumin, and C reactive protein. Clotting parameters included prothrombin time (PT), activated partial prothrombin time (APTT), and fibrinogen.

An AE was defined as any untoward medical occurrence in a clinical trial participant administered a study product, which did not necessarily have a causal relationship with this treatment. AEs were recorded throughout the study and follow-up period. All AEs were judged by the investigator with regard to severity (mild, moderate, or severe) and their relationship to the study drug (unrelated, possible, probable, or definite). Furthermore, registered events were deemed likely to be causally related when they demonstrated a temporal relationship to study drug infusion, could not be reasonably attributed to preexisting conditions, or were consistent with known pathophysiological mechanisms of infusion-related reactions. Post-hoc, AEs were subsequently mapped to standardized Medical Dictionary for Regulatory Activities (MedDRA) terms (version 27.1).

To minimize the risks for participants, dose groups were tested sequentially if the previous dose was tolerated without relevant side effects. In each dose group, the first four participants were tested consecutively, with 48 h between experimental d. An independent data safety monitoring board reviewed all safety data, including vital signs, laboratory parameters, and AEs, and approved continuation of the study with the next dose groups.

Sample collection

Ethylenediaminetetraacetic acid (EDTA) and lithium-heparin anticoagulated blood was obtained (30 min, 60 min, 90 min, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 24 h, 48 h, 72 h, Day 7, Day 14, Day 21, Day 28 after start of infusion) and centrifuged (2000 g, 4°C) within 10 min following withdrawal. Serum samples were allowed to clot for 30 min prior to being centrifuged. Plasma was stored at −80°C until analysis. For systemic adrenergic response (endogenous catecholamine concentrations) analyses, additional EDTA-anticoagulated samples were drawn at key timepoints. To prevent degradation of adrenergic metabolites during storage at −80°C, glutathione was added to the plasma after centrifugation.

PK analysis

Procizumab concentrations were measured at Eurofins ADME bioanalyses (Vergèze, France) under good laboratory practice conditions. The method was validated according to ICH Harmonised Guideline – Bioanalytical Method Validation and Study Sample Analysis M10 to quantify PCZ in human serum samples. In brief, the enzyme-linked immunosorbent assay method is based on streptavidin pre-coated microplates and 50 ng/mL biotin-DPP3 peptide to bind total PCZ molecules. Serum samples were incubated at a minimum required dilution of 20, i.e., dilution in sample buffer. After incubation and following washing steps, the immobilized drug antibody was detected with horseradish peroxidase (HRP)-conjugated mouse anti-human IgG1 Fc secondary antibody (clone HP6069). The lower limit of quantification of the assay was 320 ng/mL.

Individual and summary PK parameters were calculated using Phoenix® WinNonlin® package (Certara, version 8.4, Pharsight Corporation, Mountain View, CA, USA). A non-compartmental analysis approach consistent with i.v. administration, linear-log trapezoidal method, and uniform weighting was used for parameter estimation. These parameters include area under the concentration – time curve (AUC), maximum concentration (C_max), time to C_max (T_max), C_min (lowest observed plasma concentration), clearance (Cl), volume of distribution characterized by the terminal phase (V), apparent volume of distribution at steady state (Vss), elimination rate constant (Kel), and terminal half-life (T_½). The maximum drug concentration (C_max) and time to maximum drug concentration (T_max) were determined from observed values. Dose proportionality was evaluated descriptively by comparing dose normalized C_max and AUC_0-∞ values across dose levels, using the ratios between dose levels and the corresponding exposure parameters to assess whether systemic exposure increased in proportion to dose.

In the PK analysis, below the limit of quantitation (BLQ) concentrations were treated as zero from time zero up to the time at which the first quantifiable concentration was observed; terminal BLQ concentrations were treated as “missing.” Actual sampling times were used for all PK analyses.

PD analyses

cDPP3 concentrations were quantified in EDTA plasma samples using a cDPP3 chemiluminescence sandwich immunoassay (4TEEN4 Pharmaceuticals GmbH).²¹ The details and design principle of this assay are provided elsewhere.^14,21 cDPP3 enzyme activity was measured in EDTA plasma samples using a soluble DPP3 activity assay (SAA, 4TEEN4 Pharmaceuticals GmbH). In addition, a sequential population PK/PD model of PCZ was developed in NONMEM (version 7.5.1), comprising a two-compartment PK model and a turnover PD model describing PCZ concentration-dependent inhibition of cDPP3 activity via an Imax-IC50 function, and this PK/PD model was subsequently used for IC₅₀ estimation.

Systemic angiotensin responses were quantified in lithium-heparin plasma using equilibrium mass spectrometry analysis, reflecting overall plasmatic renin – angiotensin system (RAS) activity. For these analyses, stable isotope-labeled internal standards for angiotensin metabolites (Ang-I, Ang-II, Ang-III, Ang-IV, Ang-1–7, Ang-1–5) were added to plasma samples followed by incubation at 37°C for 1 h and quantification of angiotensin peptide levels by liquid chromatography-mass spectrometry.²² Plasma renin activity (PRA) was measured in the same samples using a liquid chromatography-mass spectroscopy approach described previously.¹³

Noradrenaline, adrenaline, and dopamine levels were measured in glutathione-stabilized EDTA plasma using routine analysis methods [high-pressure liquid chromatography with fluorometric detection, as described previously.²³

Circulating concentrations of the inflammatory cytokines tumor necrosis factor (TNF), interleukin (IL) 6, IL-8, IL-10, granulocyte-colony stimulating factor (GCSF), monocyte chemoattractant protein 1 (MCP-1), and interferon gamma-induced protein 10 (IP-10) were determined batchwise in EDTA plasma using a simultaneous Luminex assay (Milliplex, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.

Statistical analyses

Continuous variables are presented as median [interquartile range (IQR)] or mean [standard deviation (SD)], depending on distribution. Categorical variables are presented as counts and percentages. Group comparisons of continuous variables were performed using Mann–Whitney U tests, Kruskal–Wallis tests, student’s T-tests or one-way analyses of variance (ANOVA) tests, depending on the data distribution and the number of groups. Categorical data were compared using Chi-square tests. All biomarker measurements were log-transformed prior to analysis.

Between treatment-group differences over time were analyzed using two-way-RM-ANOVA tests (for normally distributed data), or two-way-RM-ANOVA tests on ranked transformed data (for non-normally distributed data). Both tests report the time*group interaction term to establish significance (implying the presence of dosage groups with differences in the parameters at a specific timepoint), which was defined as p < 0.05.

For analyses of change from baseline, the overall group (dose) effect is first reported, testing whether the average change from baseline differs across the dosage groups. When this group effect reached significance (p-value of <0.05), post-hoc comparisons were performed to assess which dosage groups were different to placebo, and in what direction. p-Values for post-hoc tests were Bonferroni corrected to account for the number of tests (n = 3). A two-sided p-value of <0.05 is considered to indicate statistical significance. All analyses were performed using R version 4.4.2 (http://www.r-project.org).

Results

Trial population

An overview of baseline characteristics of the 24 included participants is presented in Table 1. All participants received trial medication as intended, completed the follow-up and were deemed compliant with the study protocol (see CONSORT flow diagram, Supplementary Figure S1). As there was no loss to follow-up and all participants completed the trial as per protocol, no participants were excluded from any of the trial’s primary or secondary outcome analyses.

Table 1.

Baseline characteristics (mean ± sd is displayed). yGT – gamma-glutamyl transferase; APTT – activated partial thromboplastin time; ALAT – alanine aminotransferase; ASAT – aspartate aminotransferase; ALP – alkaline phosphatase; bp – blood pressure; CK – Creatinine kinase; CRP – C reactive protein; eGFR – estimated glomerular filtration rate; hf – heart frequence; LDH – lactate dehydrogenase; MAP – mean arterial pressure; PCZ – Procizumab; pt – prothrombin time; spO₂- peripheral oxygen saturation; * represent means with exclusion of participant PCZ.25 (of whom data are listed in Supplementary table S1).

	Placebo (n = 6)	PCZ 3 mg/kg (n = 6)	PCZ 6 mg/kg (n = 6)	PCZ 12 mg/kg (n = 6)
Age [years]	23.3 (1)	22 (2.4)	23.7 (3)	22.7 (2)
BMI [kg/m2]	23.2 (2.7)	24.3 (1.8)	24.4 (2.1)	23.7 (3.4)
Weight [kg]	76.3 (10.5)	84.6 (5.9)	81.2 (6.5)	79.3 (8.2)
Height [m]	1.8 (0)	1.9 (0)	1.8 (0.1)	1.8 (0)
Hemoglobin [mmol/L]	8.9 (0.3)	8.8 (0.4)	8.7 (0.5)	8.6 (0.4)
Leukocytes [count *109]	5.1 (0.9)	4.7 (0.8)	4.4 (0.5)	6.1 (1.4)
eGFR [mL/min/1.73 m2]	90 (0)	90 (0)	90 (0)	85 (12.2)
Creatinine [mmol/L]	70.8 (7.7)	78.3 (8.2)	72.5 (5.2)	73.8 (11.8)
Sodium [mmol/L]	139.3 (1.2)	139.7 (2.6)	140.2 (2)	140.8 (2.5)
Potassium [mmol/L]	3.9 (0.2)	3.9 (0.3)	3.8 (0.3)	3.6 (0.3)
PT [s]	11.6 (0.9)	11.8 (0.3)	12.1 (0.5)	12.1 (0.7)
APTT [s]	30.7 (3.3)	31.3 (2.9)	30 (1.4)	28.8 (1.5)
ASAT [U/L]*	24.7 (11.3)	21.8 (5.8)	23.3 (5.8)	20.6 (4.7)
ALAT [U/L]	21 (12.9)	18.8 (7.2)	21 (6.5)	20.5 (8)
CK [U/L]*	83.8 (28.8)	171.5 (115.2)	169.8 (158.8)	190.4 (95.4)
CRP [mg/L]	1 (0)	1 (0)	1 (0)	1.2 (0.4)
LDH [U/L]*	148.2 (12.3)	141 (13.4)	172.7 (25.2)	175 (16.7)
ALP [U/L]	69.8 (13)	64.2 (11.9)	62.3 (12.2)	57.5 (13.4)
gGT [U/L]	33.5 (44.4)	18.8 (5.8)	13.8 (4.5)	17 (3.3)
Bilirubin [U/L]	12 (6.8)	10.3 (7.3)	11.3 (8)	9.3 (3.1)
Albumin [g/L]	38.3 (2.3)	38 (2.3)	39 (1.4)	39.5 (1.9)
Thrombocytes [mmol/L]	229.5 (54.3)	196.3 (59.1)	227.8 (31.7)	210.7 (55.3)
Neutrophiles [count *109]	2.7 (0.4)	2.3 (0.6)	2.3 (0.6)	3 (1.2)
Lymphocytes [count *109]	1.7 (0.5)	1.7 (0.3)	1.6 (0.1)	2.4 (0.4)
Monocytes [count *109]	0.6 (0.2)	0.5 (0.2)	0.4 (0.1)	0.5 (0.2)
Eosinophiles [count *109]	0.2 (0.1)	0.2 (0.1)	0.1 (0)	0.2 (0.1)
Basophiles [count *109]	0 (0)	0 (0)	0 (0)	0 (0)
Hematocrit [%]	42.2 (1.6)	42.3 (1.6)	39.7 (2.3)	40.7 (1.4)
Fibrinogen [g/L]	1.9 (0.3)	2 (0.3)	2 (0.3)	2 (0.3)
Urea [mmol/L]	5.1 (1)	5.3 (1.8)	5.3 (1)	4.1 (1.2)
HF [BPM]	65.7 (12.6)	66.2 (9.5)	65.2 (6.1)	67.3 (9.2)
BP systolic [mmHg]	135.2 (18.2)	148.2 (13)	136.2 (18.6)	130.2 (6.8)
BP diastolic [mmHg]	69.5 (16.2)	75.3 (7.7)	71.8 (10)	70.5 (5.6)
MAP [mmHg]	89.7 (17.7)	90.5 (13.2)	90.8 (11.8)	89.7 (5.3)
spO2 [%]	99 (1.3)	99.7 (0.5)	98.8 (1)	99 (0.9)
Body Temperature [°C]	37.1 (0.4)	37 (0.3)	37 (0.2)	36.8 (0.7)

Safety and tolerability

Overall, PCZ was well tolerated locally and systemically across all dose levels, as demonstrated by the absence of clinically relevant or dose-dependent differences in local tolerability, vital signs, laboratory parameters, and ECGs. No deaths, serious adverse events (SAEs), or suspected unexpected serious adverse Reactions (SUSARs) occurred during the trial follow-up time.

A total of 30 AEs were recorded during the 28-d follow-up after study drug administration (Table 2). Of the 30 AE episodes, 28 were deemed to be mild, and 2 were deemed to be moderate (both occurring in the 3 mg/kg group). All AE episodes were transient and fully resolved without sequelae. At the time of occurrence, of 30 recorded AEs, 29 were categorized as “possibly related to the study drug” while only one was deemed to be “unrelated” (bicycle collision incidence). Upon unblinding and further evaluation, none were considered causally related to PCZ.

Table 2.

Overview of adverse events per treatment group (MedDRA analysis). AE = adverse event; N = number of participants per group; n = number of participants per parameter; PCZ – Procizumab. Note: percentages are based on the number of participants in each treatment group. Participants are counted once within each system organ class and preferred term.

System Organ Class Preferred Term	PCZ			Placebo	Total
	3 mg/kg N = 6 n (%)	6 mg/kg N = 6 n (%)	12 mg/kg N = 6 n (%)	N = 6 n (%)	N = 24 n (%)
Total amount of AEs	8 (27%)	12 (40%)	3 (10%)	7 (23%)	30
Participants with an AE	6 (100%)	5 (83.3%)	2 (33.3%)	4 (66.7%)	17 (70.8%)
Blood and lymphatic system disorders	0	1 (16.7%)	0	0	1 (4.2%)
Anemia	0	1 (16.7%)	0	0	1 (4.2%)
Eye disorders	1 (16.7%)	3 (50.0%)	0	1 (16.7%)	5 (20.8%)
Conjunctival hyperemia	1 (16.7%)	0	0	0	1 (4.2%)
Eye irritation	0	1 (16.7%)	0	0	1 (4.2%)
Eye pruritus	1 (16.7%)	2 (33.3%)	0	1 (16.7%)	4 (16.7%)
Gastrointestinal disorders	0	0	1 (16.7%)	0	1 (4.2%)
Diarrhea	0	0	1 (16.7%)	0	1 (4.2%)
Vomiting	0	0	1 (16.7%)	0	1 (4.2%)
General disorders and administration site conditions	2 (33.3%)	1 (16.7%)	0	2 (33.3%)	5 (20.8%)
Catheter site discomfort	0	1 (16.7%)	0	0	1 (4.2%)
Catheter site pain	1 (16.7%)	0	0	0	1 (4.2%)
Fatigue	1 (16.7%)	0	0	2 (33.3%)	3 (12.5%)
Pyrexia	1 (16.7%)	0	0	0	1 (4.2%)
Immune system disorders	1 (16.7%)	0	0	0	1 (4.2%)
Lymphadenopathy	1 (16.7%)	0	0	0	1 (4.2%)
Infections and infestations	1 (16.7%)	0	1 (16.7%)	0	2 (8.3%)
Localized infection	0	0	1 (16.7%)	0	1 (4.2%)
Tonsillitis	1 (16.7%)	0	0	0	1 (4.2%)
Injury, poisoning and procedural complications	1 (16.7%)	1 (16.7%)	0	0	2 (8.3%)
Arterial puncture site hematoma	1 (16.7%)	0	0	0	1 (4.2%)
Contusion	0	1 (16.7%)	0	0	1 (4.2%)
Musculoskeletal and connective tissue disorders	2 (33.3%)	1 (16.7%)	0	2 (33.3%)	5 (20.8%)
Knee pain	1 (16.7%)	1 (16.7%)	0	0	2 (8.3%)
Knee swelling	1 (16.7%)	0	0	0	1 (4.2%)
Musculoskeletal pain	1 (16.7%)	0	0	1 (16.7%)	2 (8.3%)
Neck pain	0	0	0	1 (16.7%)	1 (4.2%)
Nervous system disorders	0	2 (33.3%)	1 (16.7%)	2 (33.3%)	5 (20.8%)
Dizziness postural	0	1 (16.7%)	0	0	1 (4.2%)
Headache	0	1 (16.7%)	0	2 (33.3%)	3 (12.5%)
Vertigo	0	0	1 (16.7%)	0	1 (4.2%)
Respiratory, thoracic and mediastinal disorders	3 (50.0%)	4 (66.7%)	0	1 (16.7%)	8 (33.3%)
Dyspnea	0	1 (16.7%)	0	0	1 (4.2%)
Epistaxis	0	1 (16.7%)	0	0	1 (4.2%)
Nasal congestion	1 (16.7%)	2 (33.3%)	0	0	3 (12.5%)
Nasal pruritus	1 (16.7%)	0	0	0	1 (4.2%)
Pharyngitis	2 (33.3%)	0	0	1 (16.7%)	3 (12.5%)
Rhinorrhea	1 (16.7%)	2 (33.3%)	0	1 (16.7%)	4 (16.7%)

The percentage of possibly related mild AEs was comparable between the placebo group and the 3 mg/kg dose group (21%). The highest frequency of possibly related mild AEs was observed in the 6 mg/kg group (43%), while participants in the highest dose group (12 mg/kg) reported 11% of the total number of possibly related mild AEs recorded in the trial. For all AE categories, occurrence of AEs was sporadic and evenly distributed between placebo and PCZ dose groups, with no dose-response/relation being present.

Two moderate AEs were reported. One participant (PCZ.12, 3 mg/kg) experienced an exercise‑related knee distortion and abrasion 26 d post‑dose, which required ibuprofen and resolved within 5 d; the event was assessed as possibly related to study medication. Another participant (PCZ.06, 3 mg/kg) developed tonsillitis accompanied with a fever and general fatigue 20 d post‑dose, required antibiotic treatment, and fully recovered within 10 d; this event was also considered possibly related to study medication.

Overall, 17/24 participants (70.8%) had at least one MedDRA-coded AE: 13/18 (72.2%) in the combined PCZ groups and 4/6 (66.7%) in the placebo group (Table 2). The most frequently affected System Organ Classes were Respiratory, thoracic and mediastinal disorders (33.3%), Musculoskeletal and connective tissue disorders (20.8%), Nervous system disorders (20.8%), General disorders and administration site conditions (20.8%), and Eye disorders (20.8%). Typical symptoms included nasal congestion, rhinorrhea, pharyngitis, headache, and musculoskeletal pain. The MedDRA-coded dataset did not reveal any new safety signals and confirmed the absence of dose-related trends in AE frequency or severity.

In the 12 mg/kg dose group, participant PCZ.25 showed elevated CK, ASAT, and LDH levels at baseline, despite normal values at screening and otherwise normal safety laboratory parameters. These elevations fully normalized on the day of dosing and during follow‑up visits. As these aberrant values were present prior to PCZ administration, they were not considered AEs or SUSARs. All laboratory values for PCZ.25 are provided in Supplementary Table S1.

Pharmacokinetics

Circulating PCZ concentrations are presented in Figure 2, while all relevant PK parameters are summarized in Table 3. At all investigated doses, C_max was observed immediately after termination of drug infusion. Exposure in blood, expressed as the C_max and AUC_0-∞, increased in a dose-proportional manner over the dose levels 3 and 6 mg/kg, while the AUC_0-∞ was slightly higher than dose-proportional at 12 mg/kg for AUC_0-∞. Based on non-compartmental analysis, PCZ’s apparent volume of distribution during the terminal phase (V) ranged from 24.9 to 33.7 L, respectively, while the volume of distribution at steady state ranged between 9.8 and 10.6 L. Mean Cl ranged from 716 mL/h at 3 mg/kg to 478 mL/h at 12 mg/kg. The mean T_1/2 was 24.3, 34.4, and 53.1 h for the 3, 6, and 12 mg/kg group, respectively.

Figure 2.

Circulating Procizumab (PCZ) concentrations over time per treatment group shaded area represents infusion period (2 h). Base indicates start of drug infusion. The left panel displays data as mean ± sd on a segmented X-axis, the right panel displays data as mean on a continuous linear scale.

Table 3.

Mean ± sd (%CV) pharmacokinetic parameters of PCZ in the three dose groups. Data are expressed as mean ± standard deviation (%CV). AUC_0-∞, area under the plasma concentration – time curve from time zero to infinity; AUC_0-last, area under the concentration–time curve from time 0 to the time of the last measurable concentration; C_max, highest observed plasma concentration; C_min, lowest observed plasma concentration; Cl, total clearance calculated; DN_AUC_0-∞, dose-normalized AUC_0-∞; DN_AUC_0-last, dose-normalized AUC_0-last; DN_C_max, dose-normalized C_max; Kel, elimination rate constant; T_½, elimination half-life; T_last, time of last observed plasma concentration; T_max, time of highest observed plasma concentration; V, apparent volume of distribution characterized by the terminal phase; Vss, apparent volume of distribution at steady state. *median (IQR) values displayed, **AUC calculated using linear-log trapezoidal method.

PK Parameter	3 mg/kg (n = 6)	6 mg/kg (n = 6)	12 mg/kg (n = 6)
Cmax (µg/ml)	50.4 ± 6.5 (12.9)	109 ± 34.7 (31.8)	198 ± 31.5 (15.8)
DN_Cmax (µg/ml/mg)	0.199 ± 0.02 (11.9)	0.229 ± 0.09 (39)	0.208 ± 0.02 (8.3)
Cmin (µg/ml)	0.505 ± 0.167 (32.5)	0.718 ± 0.275 (38.3)	0.512 ± 140 (27.4)
AUC 0-last **(µg*h/ml)	345 ± 67.9 (19.7)	845 ± 336 (39.8)	2076 ± 584 (28.1)
DN_AUC0-last (µg*h/ml/mg)	1.35 ± 0.22 (15.9)	1.77 ± 0.81 (45.9)	2.17 ± 0.54 (24.7)
AUC0-∞ **(µg*h/ml)	363 ± 74.7 (20.6)	877 ± 339 (38.6)	2112 ± 590 (27.9)
DN_AUC0-∞ (µg*h/ml/mg)	1.43 ± 0.24 (17)	1.83 ± 0.82 (44.6)	2.21 ± 0.54 (24.6)
Tmax* (h)	2 (2–2)	2 (2–2)	2 (2–3)
Tlast* (h)	72 (72–72)	72 (72–168)	168 (168–336)
V (L)	24.87 ± 3.26 (13.1)	28.19 ± 9.94 (35.3)	33.69 ± 7.99 (23.7)
Vss (L)	10.58 ± 1.46 (13.8)	10.20 ± 3.16 (31)	9.83 ± 2.41 (24.5)
T1/2 (h)	24.3 ± 2.81 (11.6)	34.3 ± 16.6 (48.3)	53.1 ± 21.5 (40.5)
Cl (ml/h)	716.15 ± 104 (14.5)	631.02 ± 249 (39.4)	478.17 ± 127 (26.6)
Kel	0.029 ± 0.003 (10.7)	0.024 ± 0.009 (36.2)	0.016 ± 0.009 (55.8)

Pharmacodynamics

cDPP3 concentration and activity

Baseline cDPP3 concentrations were comparable in all dose groups (between 16.9 and 23.4 ng/mL, p = 0.245) and corresponded to concentrations previously reported in healthy volunteers.²⁴

cDPP3 enzyme activity at baseline was also comparable between all four groups (between 2.5 and 3.4 U/L, p = 0.220). A significant reduction in cDPP3 activity was apparent in all PCZ-treated participants, with a tendency toward a dose-dependent reduction (Figure 3, interaction p-value < 0.001). Inhibition of enzymatic activity was statistically significant from 30 min until 10 h after start of PCZ infusion for all groups (all post-hoc p < 0.001). At 24 h, a significant inhibition difference to the placebo group was still obvious for the 6 and 12 mg/kg groups (both post-hoc p < 0.01).

Figure 3.

DPP3 activity, expressed as change from baseline over time per treatment group (mean ± sd). Dotted line and error-bars represent mean ± sd (2.86 ± 0.78 U/L) DPP3 activity baseline levels, respectively (n = 24). *p-value < 0.05 for post hoc analysis (Kruskal Walis). p-value color represents corresponding dose groups.

In contrast to cDPP3 activity, no changes in cDPP3 concentrations were found after study drug administration (Supplementary Figure S2), indicating that only enzyme activity, not concentration, was modulated by PCZ administration.

The relationship between circulating PCZ concentrations and cDPP3 enzyme activity is visualized in Figure 4(A–D). For each dose group, a clear relation between PCZ concentrations falling below the experimentally established IC₅₀ of 3.85 µg/mL (indicated by the dotted line in the figure panels) and recovery of enzyme function can be observed.

Figure 4.

DPP3 activity, expressed as change from baseline (U/L) and corresponding Procizumab (PCZ) concentration level (µg/mL) for different dose groups (mean ± sd): panel A: 3 mg/kg panel B: 6 mg/kg, and panel C: 12 mg/kg (n = 6 per dose group), panel D: all dose groups together. Dotted line indicates PCZ’s IC₅₀ (corresponding to 3.85 µg/mL as determined based on a PKPD model and confirmed by experimental in vitro data).

The data in Figures 3 and 4 are presented using a segmented (categorical) X-axis to enhance visualization of key timepoints. For clarity, the same data is plotted using a continuous linear scale in supplementary figure S3 and supplementary figure S4.

Systemic angiotensin responses

Angiotensin I, II, III, IV concentrations as well as plasma renin activity (PRA) activity increased over time in all dose groups and placebo in comparison to the respective baseline values, with no dose-dependent effects observed (Figure 5, all p < 0.001 for factor time and all p > 0.05 for interaction with dose).

Figure 5.

Change in angiotensin I, angiotensin ii, angiotensin iii, angiotensin IV, and plasma renin activity compared to baseline at 2 h, 10 h, and Day 7 after placebo or Procizumab (PCZ) administration at 3, 6, and 12 mg/kg. *p-value < 0.05 against placebo for post hoc analysis. Ang = angiotensin, PRA = plasma renin activity.

Post-hoc test results for change of angiotensins I, II, III, IV, and PRA at individual timepoints suggested increased concentrations/activity at the 2 h post-PCZ start time point in the 3 mg/kg and 12 mg/kg dose groups compared to placebo (Figure 5). However, because these results did not reach statistical significance at later timepoints and were not found for the 6 mg/kg subgroup, a clear dose-dependency of these effects was not established.

Systemic adrenergic responses

No significant changes from baseline were observed for any of the assessed adrenergic metabolites (Supplementary Figure S5, all p > 0.120). Furthermore, post-hoc results for individual timepoints also did not show significant differences for any timepoint between any dose group (Supplementary Figure S5).

Discussion

We evaluated the safety, tolerability, and PK/PD of single escalating i.v. doses of PCZ in healthy male volunteers. PCZ exhibited an excellent safety profile, demonstrated by local tolerability, vital signs, laboratory parameters, and ECGs. No SAEs occurred during the trial. In all three dose groups, the C_max was attained at or shortly after termination of infusion. Dose-dependent increases in C_max and AUC_0–∞ were observed. A low volume of distribution indicates that PCZ predominantly distributes over plasma.

Safety

All AEs reported for this study were transient and fully resolved during the study’s follow-up period. For any of the adverse event categories reported, no dose-dependency was observed. The absence of dose-dependency was also noted for respiratory tract symptoms (i.e., itchy and runny nose, nasal congestion), the most prevalent recorded adverse event for this trial.

All AEs related to respiratory tract symptoms occurred in the placebo, 3 mg/kg and 6 mg/kg group, while none were recorded for the 12 mg/kg dose group. The uneven distribution of this AE category (with no clear dose–response relationship) is likely explained by the season during which this trial was performed. Seasonal allergic rhinitis, commonly known as hay fever, affects approximately 20 to 30% of the population in north-western Europe, including the Netherlands.²⁵ In the Netherlands, the hay fever season typically begins in early spring, at or around the start of April. Both the 3 and 6 mg/kg dose groups (and half of the placebo subjects) coincided with the peak hay-fever incidence of Northern Europe. As respiratory tract-related symptoms were not present in the highest dose group, a dose-dependent exacerbation of allergy-related complaints caused by PCZ appears unlikely.

Pharmacokinetics

PCZ was cleared from the circulation relatively fast (T_1/2= ~1–2 d) in comparison to other monoclonal antibodies in clinical use (between 11–30 d) and endogenous IgG antibodies (~21 d).^26,27 The relatively short half-life observed across all dose groups may have been influenced by the sensitivity of the applied bioanalytical method. With a more sensitive method, measurable concentrations might have been detected at more timepoints, which impacts half-life calculations.

Alternatively, it could be a characteristic of the antibody or caused by target-mediated clearance, with PCZ-DPP3 complexes tending toward rapid clearance kinetics, similar to what has been described for DPP3.^28,29 DPP3, while being a large protein (~82–83 kDa), has a short circulating half-life of approximately 20–70 min.^12,14 Although the mechanisms responsible for cDPP3’s rapid clearance from the circulation have not been investigated in detail, studies on the clearance kinetics of other enzymes suggests endocytosis in the liver followed by further processing in lysosomes.³⁰ Based on the relatively short half-life values compared to other IgG antibodies, the rapid clearance of PCZ-DPP3 complexes from the circulation appears likely. Corresponding with this observation, PK data indicates that PCZ may exhibit a non-linear, saturable kinetic with higher doses resulting in a longer half-life, slower clearance, and slightly higher than dose-proportional blood exposure. This phenomenon, where the interaction between an antibody and its intended target contributes to the antibodies clearance rate, is called target-mediated drug disposition (TMDD).²⁸ At low PCZ concentrations (i.e., subtherapeutic doses), TMDD could contribute to a significant proportion of PCZ clearance. With increasing PCZ dose and concentration, the clearance decreases potentially because the TMDD pathway becomes saturated and the fraction of unbound PCZ increases. Therefore, at higher PCZ concentrations, the linear clearance pathway could be more prominent. This phenomenon of biphasic clearance versus the dose profile (non-linear and linear clearance) was already described for several therapeutic monoclonal antibodies, including cetuximab, trastuzumab, and efalizumab.^27,31,32

Although the observed dose-related decrease in clearance and prolongation of half-life are broadly compatible with TMDD, several unrelated alternative mechanisms could also explain the observed PK behavior. First, hepatic mannose receptor-mediated uptake could potentially clear PCZ more efficiently at lower concentrations, but become saturated at higher doses, resulting in reduced clearance and longer half-life.³³ Second, formation of DPP3–PCZ complexes may impair neonatal Fc receptor (FcRn) binding, putatively reducing antibody recycling efficiency. At higher doses, a larger fraction of free, unbound PCZ remains available for FcRn-mediated recycling, which may further contribute to the dose‑dependent prolongation of half-life.

Given that this study evaluated only three single-dose levels in healthy volunteers, with low endogenous cDPP3 and relatively sparse late-timepoint sampling, all proposed mechanisms explaining PCZ’s PK behavior should be regarded as plausible working hypotheses rather than definitive explanations at this stage.

Of note, these results may indicate that the rate of PCZ elimination could be enhanced if higher cDPP3 concentrations are present in the circulation, as occurs in patients during cardiogenic shock.¹¹ Consequently, the influence of varying pathophysiological cDPP3 concentrations on PCZ’s half-life warrants further investigation in Phase 1b clinical safety studies. Based on the results of this study, the multicenter PROCARD Phase 1b/2a safety and PK study is currently ongoing in cardiogenic shock patients (ClinicalTrials.gov NCT06832722).

Anti-drug antibody (ADA) immunogenicity testing was not performed in this study, as PCZ’s single-dose administration, suspected rapid clearance, and transient pharmacological effect made any ADA-related impact on PK/PD parameters or safety unlikely. Regardless, ADA immunogenicity testing will be incorporated into the ongoing PROCARD 1b/2a trial. This study’s relatively small cohort size (8 participants per dose level), though in line with common first-in-human study design,^34,35 limited the precision of our PK variability estimates. However, as the observed variability was within the typical range for early-phase trials, this limitation did not preclude qualitative conclusions on dose-proportionality and the overall PK profile of PCZ.

Pharmacodynamics

The main PD effect of PCZ serum concentrations on cDPP3 enzyme activity inhibition demonstrated similar profiles to those observed in previously performed animal disease models.^12,17,19 A clear inhibitory effect of PCZ could be observed on cDPP3 activity while the antibody concentration was above the IC₅₀, despite the lower cDPP3 levels in healthy humans compared to previous experiments in animal disease models ^12,17,19 and diseased patients.^10,36 Depending on the dose, PCZ administration provided relevant inhibition of cDPP3 activity up to 10 h (3 mg/kg subgroup) and 24 h (6 and 12 mg/kg subgroups) after study drug administration. PCZ did not significantly impact cDPP3 plasma concentrations. These results suggest good translatability of PCZ’s mode of action from animals to humans.

PCZ administration did not result in relevant changes in blood pressure in healthy volunteers at any of the assessed doses. Based on previous studies in healthy animals, relevant hemodynamic effects were not expected, since inhibition of low physiological cDPP3 activity in healthy animals did not affect systemic hemodynamic parameters as well.^12,18 While we did find an increase of circulating angiotensin I, II, III, IV concentrations, and PRA after study drug administration, this is unlikely caused by a direct effect of PCZ infusion. Based on PCZ’s proposed mode of action,⁹ cDPP3 inhibition prevents the decay of hormonally active RAAS peptides. Indeed, PCZ administration resulted in increased concentrations of angiotensin II, 1–5 and 1–7 in healthy mice, while PRA and the hormonally inactive precursor angiotensin I were not affected.¹⁸ Considering that all RAAS metabolites assessed in the current study followed the same pattern as PRA, our data suggests that all changes in RAAS metabolites were driven by increases in PRA, rather than inhibition of the low constitutive cDPP3 activity known to be present in a healthy state. Correspondingly, inhibition of cDPP3 activity also did not significantly affect catecholamine concentrations. Given the small sample size and the high RAAS metabolite variability, this study was likely underpowered to detect transient effects of cDPP3 inhibition. Moreover, in the absence of shock, RAAS activity is low, Angiotensin II is not elevated, and circulating DPP3 concentrations are normal. This physiological context likely serves as an additional explanation, as this also reduced the effects of detectable RAAS modulation by PCZ. To what extent PCZ administration affects RAAS metabolite function and hemodynamic parameters in humans when high cDPP3 concentrations are present (i.e., during cardiogenic shock) should be investigated in follow-up human studies. The most recent preclinical study performed in pigs with septic shock demonstrated that PCZ administration increased angiotensin II, III, IV, and 1–5 concentrations and reduced both fluid and catecholamine requirements.¹⁹

Conclusion

PCZ was safe and well tolerated in male, healthy adult participants when administered as a single i.v. infusion (3, 6, or 12 mg/kg) over 2 h. Furthermore, it potently reduced cDPP3 activity for 10–24 h, depending on the administered dose. The results of this study are followed up in the currently ongoing Phase 1b safety and PK study (PROCARD 1b/2a) in patients with cardiogenic shock and elevated cDPP3 concentrations. PROCARD 1b/2a aims to establish the optimal dose of PCZ to be implemented in a subsequently planned efficacy study.

Funding Statement

This study was funded by 4TEEN4 Pharmaceuticals GmbH.

Disclosure statement

DvL and MK were invited to a meeting in Berlin by 4TEEN4 Pharmaceuticals GmbH. PP received travel and consultancy reimbursements from 4TEEN4 Pharmaceuticals GmbH. KS is employed by 4TEEN4 Pharmaceuticals GmbH, the company holding the patent rights for the Procizumab compound. AB is the CEO of 4TEEN4 pharmaceuticals. All other authors have nothing to declare.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2026.2671468