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. 2021 Dec 21;5(1):41–51. doi: 10.1021/acsptsci.1c00227

In Vivo Pharmacodynamic Method to Assess Complement C5a Receptor Antagonist Efficacy

Cedric S Cui †, Vinod Kumar †, Declan M Gorman †, Richard J Clark †, John D Lee †, Trent M Woodruff †,‡,*
PMCID: PMC8762733  PMID: 35059568

Abstract

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The complement C5a receptor 1 (C5aR1) has been studied as a potential therapeutic target for autoimmune and inflammatory diseases, with several drug candidates identified. Understanding the pharmacokinetics and pharmacodynamics of a drug candidate is a crucial preclinical step that allows for a greater understanding of a compound’s in vivo biodistribution and target engagement to assist in clinical dose selection and dosing frequency. However, few in vivo pharmacodynamic methods have been described for C5a inhibitors. In this study, we, therefore, developed a complete in vivo pharmacodynamic assay in mice and applied this method to the peptide-based C5aR1 antagonists PMX53 and JPE-1375. Intravenous administration of recombinant mouse C5a induced rapid neutrophil mobilization and plasma TNF elevation over a 60 min period. By using C5a receptor-deficient mice, we demonstrated that this response was driven primarily through C5aR1. We next identified using this model that both PMX53 and JPE-1375 have similar in vivo working doses that can inhibit C5aR1-mediated neutrophilia and cytokine production in a dose as low as 1 mg/kg following intravenous injection. However, the in vivo active duration for PMX53 lasted for up to 6 h, significantly longer than that for JPE-1375 (<2 h). Pharmacokinetic analysis demonstrated rapid plasma distribution and elimination of both compounds, although PMX53 had a longer half-life, which allowed for the development of an accurate pharmacokinetic/pharmacodynamic model. Overall, our study developed a robust in vivo pharmacodynamic model for C5aR1 inhibitors in mice that may assist in preclinical translational studies of therapeutic drug candidates targeting C5a and its receptors.

Keywords: pharmacodynamics, C5a, C5a receptors, C5aR antagonist, neutrophils, TNF

The complement system plays a vital role in defending against invading pathogens and in many biological processes.1 All complement activation pathways lead to the cleavage of the complement factor C5, generating C5a. This complement peptide binds its G protein-coupled seven transmembrane spanning receptor, C5a receptor 1 (C5aR1), and induces tissue inflammation and recruitment of immune cells.2 C5a also binds with equal potency to a second seven transmembrane spanning receptor, C5aR2, although due to lack of G-protein coupling, exerts more nuanced functions.3 In recent decades, many inflammatory and neuroinflammatory disorders have been identified to be driven, in part, through the unregulated activation of the C5a–C5aR1 axis.4−7 Therefore, inhibition of C5aR1 is viewed as a promising disease-modifying therapeutic strategy, and several small molecules, antibodies, and peptide-based C5aR1 antagonists have been developed. The peptide-based C5aR1 inhibitors, however, have shown particular utility due to their ability to be used across many species, including rodents, which has generated abundant knowledge of C5a’s role in biology and pathology.

The first peptide-based C5aR1 antagonists described were principally developed by modifying the C-terminal region of C5a. This led to the first full C5aR1 antagonist, the hexapeptide, MeFKPdChaWr.8 This compound then spurred the discovery of further antagonists targeting C5aR1, including PMX53 (also referred to as 3D53),9 PMX205,4,10 and JPE-1375.11 PMX53 (AcF-[OPdChaWR]) was first developed over 20 years ago. It is a potent cyclic hexapeptide C5a receptor antagonist with an IC50 of 20 nM on intact human polymorphonuclear leukocytes (PMNs).9 It has been shown to have therapeutic effects in several animal models, including sepsis,12 inflammatory bowel disease,13,14 diabetic kidney disease,15 ischemia-reperfusion injuries,16−19 spinal cord injury,20 and neurodegenerative disease.4

In contrast to the cyclic peptide analogues (i.e., PMX53 and PMX205), JPE-1375 (Hoo-Phe-Orn-Pro-hle-Pff-Phe-NH2) is a linear peptidomimetic C5aR1 antagonist.11 This peptide replaces the C-terminal Arg of PMX53 with a hydrophobic amino acid, which was demonstrated to retain C5aR1-binding affinity and increase the stability and specificity toward C5aR1. In vivo studies also indicated improved bioactivity in a mouse model of the reverse passive arthus reaction (RPAR),11 although the finding of increased potency over the PMX analogues has not been independently replicated.

The in vitro pharmacological characterization of both PMX53 and JPE-1375 has been recently compared in human cells, which documented their potencies and antagonistic activities in a variety of signaling and functional assays.21 However, it is also important to determine the in vivo pharmacological profile of these compounds to assist researchers in identifying the appropriate drug concentration and dose frequency for future preclinical studies and assist in a clinical dose selection of related inhibitors. A pharmacokinetic and in vitro to in vivo extrapolation stability study of cyclic peptide C5aR1 antagonists using LC–MS has been developed.22 However, no in vivo pharmacodynamic study has been performed to compare cyclic peptides such as PMX53 and the linear peptide JPE-1375. A pharmacodynamic study will, therefore, help to understand the working dose(s) required to inhibit C5aR1 in vivo, and the period of in vivo C5aR1 inhibition.

In the current study, we established a pharmacodynamic assay by measuring C5a-induced neutrophil mobilization and TNF generation in mice and determined the efficacy of the C5aR1 antagonists PMX53 and JPE-1375. We demonstrated that both PMX53 and JPE-1375 inhibited C5a-induced neutrophil mobilization at an intravenous dose of 1 mg/kg. PMX53’s activity on neutrophil mobilization lasted for 6 h and JPE-1375 for 2 h, which correlated with the longer pharmacokinetic half-life of PMX53, allowing for an accurate pharmacokinetic/pharmacodynamics (PK/PD) profile to be generated for these compounds.

Materials and Methods

Animals

C57BL/6J wild-type (WT) and C5aR1–/–23 male mice at 10–12 weeks of age were used for this study. The breeding colony for all the animals was maintained at the University of Queensland Biological Resources Animal Facilities. Animals were group-housed (4 animals per cage) under identical conditions in a 12 h light/dark cycle (lights on at 06:00 h) with free access to food and water. All animal procedures were approved by the University of Queensland Animal Ethics Committee and conducted under the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th Edition, 2013).

Materials and Reagents

The peptide C5aR1 antagonists PMX53 and JPE-1375 were synthesized in-house as previously described.21 All compounds were validated at >95% purity as determined using LC–MS/MS. Endotoxin-free recombinant mouse C5a (Cat# 51136-MNAE) was obtained from the Sino Biologicals (Beijing, China). Hemacolor Rapid Staining of Blood Smear Kit (Cat#111661) and dibutylphthalate polystyrene xylene (DPX) (Cat# 1.00579.0500) were brought from Merck (Darmstadt, Germany). Mouse TNF ELISA set II (Cat#558534) was obtained from BD Biosciences (San Diego, CA).

Experimental Protocol

PMN Mobilization Assay

WT or C5aR1–/– mice on a C57BL/6J genetic background (n = 4/group) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg/kg via intravenous (i.v.) injection (via the tail vein). One drop of blood was collected from the tail tip, and a blood smear performed on a slide at 0, 15, 30, and 60 min after C5a injection. Blood smears were then stained using Microscopy Hemacolor Rapid Staining of Blood Smear Kit (Merck, Germany). Briefly, blood smears were fixed in Hemacolor Solution 1 (methanol). The slides were then stained with Hemacolor Solution 2 (Eosin Y), followed by Hemacolor Solution 3 (Azur B). Slides were washed with 1× phosphate-buffered saline (PBS) (pH 7.2) and mounted with dibutyl phthalate polystyrene xylene. Using the 20×/0.4 NA objective on an Olympus CX21 microscope, the first 200 white blood cells were counted, and the proportion (%) of PMNs (cells with clear granules that are light violet in color) was calculated.

Dose Determination

C5aR1 antagonists PMX53 and JPE-1375 were administered to WT mice via the i.v. route at 0.3, 1.0, and 3.0 mg/kg. 15 min following PMX53 and JPE-1375 administration, recombinant C5a protein at 50 μg/kg was injected i.v. and PMN mobilization and TNF production assessed at 60 min (Figure 1A).

Figure 1.

Figure 1

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Schematic of PMN mobilization assay using C5aR1 antagonists JPE-1375 and PMX53.

Time Profile

To determine the bioactivity time profile of the C5aR1 antagonists PMX53 and JPE-1375, drugs were administered to WT mice i.v. at 0.25 2, 6, and 24 h prior to the i.v. injection of recombinant C5a protein at 50 μg/kg. PMN mobilization and TNF production were then determined 60 min after C5a injection (Figure 1B).

Enzyme-Linked Immunosorbent Assay

Blood at terminal time points for all experiment groups was collected from the inferior vena cava in 4 mM ethylenediaminetetraacetate (EDTA), and plasma obtained by centrifugation for 10 min at 2000g at 4 °C. Plasma TNF levels were determined using a commercially available enzyme-linked immunosorbent assay kit following the manufacture’s protocol (BD Biosciences).

Flow Cytometry

Flow cytometry was performed to identify and quantify PMN/neutrophil populations in blood samples of C5a-treated WT mice at 0 and 60 min. Blood samples (100 μL) were collected from mice through tail bleeds. The samples were diluted 1:4 with anticoagulant buffer (4 mM EDTA in 1× PBS, pH 7.4) and subsequently lysed with red blood cell lysis buffer (0.85% NH4Cl in ddH2O, pH 7.4) for 5 min at room temperature. Following centrifugation at 500g for 5 min at 4 °C, the cells were resuspended in flow cytometry blocking buffer (0.5% BSA, 2 mM EDTA in 1× PBS, pH 7.4), followed by incubation with rat anti-CD16/32 (1:200; BD Biosciences) for 10 min at 4 °C to block FC receptors. Cells were immunolabeled with rat anti-Ly6G-BUV395 (1:100; clone:1A8; BioLegend) and rat anti-CD11b-PE (1:400; clone: M1/70; BioLegend) for 1 h at room temperature. All antibodies were diluted in the blocking buffer (0.5% BSA, 2 mM EDTA in 1× PBS, pH 7.4). For the exclusion of dead cells, samples were also incubated with near infrared conjugated viability dye Zombie NIR (1:100; BioLegend). Stained samples were then analyzed using an LSR II flow cytometer (BD Biosciences) and FlowJo analysis software. Cell doublets and triplets were excluded based on FSC-A/FSC-H linearity.

Pharmacokinetic and Biodistribution Studies

The plasma pharmacokinetic profiles of PMX53 and JPE-1375 were assessed in a separate group of male mice (n = 5), following i.v. administration at a dose of 1 mg/kg. Serial blood samples were collected at 10, 15, 30, 45, and 60 min, and at 2, 4, 6, and 24 h via a tail vein using a microsampling technique.24 At the terminal time point, that is, 24 h, animals were anesthetized with zolazepam (50 mg/kg) and xylazine (12 mg/kg) via i.p. injection. Blood samples were collected from cardiac puncture as well as from tail bleed to validate the sampling protocol. All blood samples were centrifuged at 2000g for 10 min at 4 °C, and the separated plasma samples were collected and stored at −80 °C for further analysis. On the day of analysis, aliquots (5 μL) of thawed plasma samples were mixed with internal standard (10 μL) and processed for peptide quantification using the validated LC–MS/MS method as described previously.22 The LC–MS/MS system consisted of an API 3200 (AB SCIEX) triple quadrupole LC–MS/MS mass spectrometer containing a Turbo V electrospray ionization source system united with a Genius AB-3G nitrogen gas generator (Peak Scientific). The mass spectrometer was operated in multiple reaction monitoring scan mode for identification of fragments in positive ionization mode at unit resolution. Compound-dependent and source-dependent mass spectrometer conditions were optimized using automatic optimization by infusion method for PMX53, JPE-1375, and internal standard (FOB) (Table S1). For quantitative purposes, one mass/charge (m/z) transition per analyte was monitored. Additional transitions were monitored for qualification purposes. For all analytes, the ion spray voltage and source temperature/auxiliary gas temperature were set at 5500 V and 500 °C, respectively. Nebulizer gas (ion source gas 1, GS1) and auxiliary gas (ion source gas 2, GS2) were set at 45 psi. The curtain and collision-activated dissociation gases were set at 30 and 5 on an arbitrary scale. The mass spectrometer was coupled with an Agilent 1200 series HPLC system (Agilent Technologies) equipped with a degasser, a column oven, a binary pump, and a temperature-controlled autosampler. The autosampler was set at 4 °C, and column oven temperature was maintained in the range of 30 ± 1 °C. The system control and data acquisition were executed by Analyst software (AB SCIEX, Applied Biosystems Inc., USA, version 1.5.1). Chromatographic separation was implemented using the Kinetex EVO C18 analytical column (100 × 2.1 mm, 100 Å, 5 μm, Phenomenex Inc., CA, USA) under binary gradient conditions using mobile phase A (0.1% formic acid in LC grade Milli-Q water pH 3) and mobile phase B (0.1% formic acid in 100% acetonitrile solvent) with a 350 μL/min flow rate. Analytes were eluted using the binary gradient, that is, 2% mobile phase B from 0 to 1 min with a linear increase to 85% from 1 to 2 min, followed by a linear increase to 98% till 3 min, hold at 98% up till 8 min. Column equilibration was achieved by a linear decrease to 1% from 8 to 8.5 min, followed by 2% of mobile phase B for the last 1 min. An injection volume of 10 μL was set for method development and sample analysis. The concentrations of the peptide in samples were measured against the known concentration of the internal standard and peak area ratio of the analyte to the internal standard.

Statistical Analysis

Data from each group were combined and expressed as mean ± standard error of the mean (SEM) unless otherwise described. All analyses were performed using GraphPad Prism 9.2.0 (San Diego, CA, USA). The statistical difference of both the percentages of circulation PMNs and plasma TNF levels was determined by ordinary one-way ANOVA with Dunnett’s multiple comparison test as indicated. For dose–response and time profile studies, the inhibition curves were normalized by the non-linear fit of dose or length of dosing time versus percentage of maximal inhibition. Values of P < 0.05 were regarded as statistically significant, and significance is presented as *, P < 0.05; **, P < 0.01; ***, P < 0.001, and ****, P < 0.0001. The Pearson correlation coefficient was used to access the relationship between the drug in the plasma concentration, % inhibition of PMNs, and % inhibition of TNF in the pharmacokinetic/pharmacodynamics models. A strong positive correlation was assumed as r ≥ 0.8, a moderate positive correlation as 0.5 ≤ r < 0.8, and a weak positive correlation as 0.3 ≤ r < 0.5. LC–MS/MS data processing and analysis were performed using Analyst software (AB SCIEX, Applied Biosystems Inc., USA, version 1.5.1) and Multiquant software (AB SCIEX, USA, version 2.0). Pharmacokinetics data analysis was performed using Phoenix WinNonlin software using non-compartmental analysis (Certara, L.P, St. Louis, Missouri, USA, v8.1). The linear trapezoidal rule was applied for the calculation of area under the curve (AUC 0–t) of the plasma concentration versus time profiles.

Results

C5a-Induced PMN Mobilization and TNF Production is Reduced in the Absence of C5aR1 Signaling in Mice

It was previously established that the complement C5a can trigger PMN mobilization (predominantly neutrophils) from the bone marrow reservoirs to the circulation in mice.38 We confirmed these findings by demonstrating increases in circulating PMN numbers from around 10% at baseline, to 60% 60 min following recombinant mouse C5a (50 μg/kg, i.v.) injection (Figure 2A,B). This experiment also identified that sterile saline-injection alone caused a progressive rise in circulating neutrophils over 60 min (from 10 to 20% of total leukocytes) (Figure 2A), similar to findings in previous studies.25,26 We further confirmed the C5a-mediated mobilization response finding by flow cytometry, where neutrophils (CD11b++Ly6G++ cells) at baseline were 9.8% of total leukocytes (1.25 ± 0.24 × 106 blood neutrophils), which then markedly increased up to 60.8% (10.4 ± 0.25 × 106 blood neutrophils) 60 min after C5a i.v. injection (Figure 2C,D).

Figure 2.

Figure 2

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C5a induces PMN mobilization and TNF elevation via C5aR1 in mice. WT or C5aR1–/– mice were injected intravenously with recombinant mouse C5a (50 μg/kg) and blood collected and assessed for leukocyte numbers and TNF. (A) Percentage of circulating PMNs after saline or C5a injection at 0 15, 30, and 60 min (n = 4). (B) Microscope view of stained blood smear slides before C5a injection and 60 min after C5a injection; red arrows and the inset show PMNs. (C) Representative flow cytometry dot plot showing the percentage of neutrophils (CD11b++, Ly6G++ cells) in total white blood cells at 0 and 60 min after C5a injection. (D) Number of neutrophils (CD11b++, Ly6G++ cells) in mouse blood 0 and 60 min after C5a injection (n = 4). (E) Percentage of circulating PMNs in WT and C5aR1–/– mice at 60 min after saline or C5a injection (n = 4). (F) Plasma concentration of TNF in WT and C5aR1–/– mice 60 min after C5a injection (n = 4). Data represent mean ± SEM. Statistical analyses were performed using either a one-way ANOVA with Dunnett’s multiple comparisons or a two-way ANOVA with Tukey’s multiple comparison test (*p < 0.01; ***p < 0.005; and ****p < 0.0001).

To investigate the contribution of C5aR1 to C5a-mediated PMN mobilization, we next compared mobilization responses between WT and C5aR1–/– mice. We found that while C5a increased blood PMNs to ∼60% in WT mice at 60 min, C5a injection in C5aR1–/– mice only increased circulating PMNs to around 30% (Figure 2E). Notably, C5a-mediated PMN mobilization was not completely attenuated in the absence of C5aR1 (compared to the 20% baseline in saline-injected WT mice), confirming a role for C5aR2 in neutrophil mobilization as recently documented.19,27

It is also well known that C5a signaling induces proinflammatory cytokine release in vivo, in particular TNF, which is involved in acute phase responses.28,29 Therefore, we tested plasma TNF levels 60 min after C5a injection. TNF increased by over 5-fold following C5a injection when compared to saline, and this response was significantly blunted in C5aR1–/– mice (Figure 2F).

In Vivo Efficacy Dose Determination of PMX53 and JPE-1375 in the C5a Pharmacodynamic Model

The pharmacodynamic study of two C5aR1 antagonists, cyclic peptide PMX53 and linear peptide JPE-1375, was next performed using C5a-induced PMN mobilization and TNF production at 60 min as output assays. Initially, we performed a dose–response study of PMX53 and JPE-1375 ranging from 0.3 to 3 mg/kg (refer to Figure 1A); doses were chosen based on prior studies using these dose ranges in therapeutic experiments in mice.11,30,31 We demonstrated that both PMX53 and JPE-1375 significantly decreased C5a-mediated PMN mobilization at 1 and 3 mg/kg doses, while no effect was observed at a 0.3 mg/kg dose (Figure 3A,B). No significant differences in the efficacy or potency of both compounds were observed in this assay, with a similar median effective concentration (EC50) for PMX53 (7.7 μM) and JPE-1375 (6.9 μM) calculated (Figure 3C). Similar to PMN mobilization, PMX53 and JPE-1375 also showed a significant reduction in TNF plasma levels at 1 and 3 mg/kg dose with both compounds reducing C5a-mediated TNF by about 90% (Figure 3D,E). At the 0.3 mg/kg dose, PMX53 and JPE-1375 only partially reduced TNF levels (Figure 3D,E). The inhibition of TNF levels also presented a similar median effective concentration (EC50) for PMX53 (5.9 μM) and JPE-1375 (4.5 μM) (Figure 3F).

Figure 3.

Figure 3

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Dose–response of PMX53 and JPE-1375 through i.v. injections in vivo. WT mice were intravenously pretreated with different doses of PMX53 or JPE-1375, and pharmacological inhibition of C5aR1 was measured with blood leukocyte numbers and TNF. (A) Percentage of circulating PMNs after intravenous injection of PMX53 (0.3, 1, and 3 mg/kg, i.v.) and 50 μg/kg C5a (n = 4). (B) Percentage of circulating PMNs after intravenous injection of JPE-1375 (0.3, 1, and 3 mg/kg, i.v.) and 50 μg/kg C5a (n = 4). (C) The associations between inhibition of PMN mobilization and dose of PMX53 or JPE-1375 by i.v. dosing. Median effective concentration (EC50) of PMX53 is 7.7 μM; EC50 of JPE-1375 is 6.9 μM (n = 4). (D) Plasma concentrations of TNF induced by C5a in WT mice pretreated with different doses of PMX53 (0.3, 1, and 3 mg/kg, i.v.). (E) Plasma concentration of TNF induced by C5a in WT mice pretreated with different doses of JPE-1375 (0.3, 1, and 3 mg/kg, i.v.). (F) Associations between inhibition of TNF and dosage of PMX53 or JPE-1375 (i.v.). EC50 of PMX53 is 5.9 μM; EC50 of JPE-1375 is 4.5 μM. Molar doses of compounds were calculated from the administration dose divided by estimated mouse blood volume (2 ml). Data represent mean ± SEM. Statistical analyses were performed using Nonlin fit and a one-way ANOVA with Dunnett’s multiple comparison test (****, p < 0.0001).

In Vivo Time Profile of PMX53 and JPE-1375 in the C5a Pharmacodynamic Model

After determining the effective dose of PMX53 and JPE-1375, we next aimed to investigate the period of active inhibition for these C5aR1 antagonists using our established pharmacodynamic model. We thus compared the efficacy of PMX53 and JPE-1375 by administering a 1 mg/kg i.v. dose at different time points (i.e., 0.25, 2, 6, and 24 h) before C5a i.v. injection (refer to Figure 1B). We identified that PMX53 significantly inhibited C5a-mediated PMN mobilization at 0.25, 2, and 6 h (Figure 4A), while JPE-1375 only decreased PMN mobilization when administered 0.25 h prior to C5a (Figure 4B). Normalizing the responses of PMX53 and JPE-1375 to C5a-induced PMN mobilization, we demonstrated a longer median effective time (ET50) of 14.0 h for PMX53, compared with ET50 of 1.3 h for JPE-1375 (Figure 4C). PMX53 also significantly decreased C5a-induced TNF levels by ∼90% at 0.25, 2, and 6 h (Figure 4D), while JPE-1375 only decreased TNFα levels by this degree (∼90%) at 0.25 h (Figure 4E). Comparing drug responses to TNF inhibition identified an ET50 of 15.1 h for PMX53 and 5.3 h for JPE-1375 (Figure 4F). Taken together, these pharmacodynamic data demonstrate that PMX53 exerts efficacy in vivo for a longer time period than JPE-1375.

Figure 4.

Figure 4

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In vivo time profile of C5aR1 antagonist PMX53 and JPE-1375 through 1 mg/kg i.v. injections. WT mice were intravenously pretreated with 1 mg/kg of PMX53 or JPE-1375 at 15 min, 2 h, 6 h, and 24 h before C5a injection (50 μg/kg, i.v.), and pharmacological inhibition of C5aR1 as measured with blood leukocyte numbers and TNF. (A,B) Percentage of circulating PMNs after intravenous injection of 50 μg/kg C5a (n = 4), and mice were intravenously pretreated with 1 mg/kg of (A) PMX53 or (B) JPE-1375 at 15 min, 2 h, 6 h, and 24 h before C5a injection. (C) Association between inhibition of PMN mobilization and period after injection of PMX53 or JPE-1375 1 mg/kg by i.v. injection. The median effective time (ET50) of PMX53 is 14.0 h, and the ET50 of JPE-1375 is 1.3 h. (D,E) Plasma concentration of TNF in WT mice intravenously pretreated with 1 mg/kg (D) PMX53 or (E) JPE-1375 at 15 min, 2 h, 6 h, and 24 h before C5a injections (50 μg/kg, i.v.). (F) Associations between the inhibition of TNF and the duration after PMX53 or JPE-1375 by 1 mg/kg i.v. The median effective time (ET50) of PMX53 is 15.1 h, ET50 of JPE-1375 is 5.3 h. Statistical analyses were performed using a Nonlin fit and one-way ANOVA with Dunnett’s multiple comparison test (***, p < 0.001; ****, p < 0.0001).

Pharmacokinetic/Pharmacodynamic Relationship of PMX53 and JPE-1375 in Mice

As a significant difference in the effective time period was observed between PMX53 and JPE-1375, we next questioned whether this could be driven purely by the different pharmacokinetic profiles of the compounds. For example, an increased plasma stability and half-life of PMX53 due to its cyclic nature could allow for a greater period of drug exposure to C5aR1-bearing cells. Alternatively, an increased receptor residence time and the non-competitive pharmacological nature of the cyclic peptide could also be driving this differential activity.10,21,32 Therefore, in order to develop a pharmacokinetic/pharmacodynamic model, we determined the pharmacokinetic profiles for PMX53 and JPE-1375. The mean plasma drug concentration versus time profiles of PMX53 and JPE-1375 following an i.v. bolus dose of 1 mg/kg is shown in Figure 5A, and the corresponding pharmacokinetic parameters as determined by non-compartmental analysis using Phoenix WINNONLIN software are summarized in Table 1. Both PMX53 and JPE-1375 demonstrated a rapid distribution in the plasma, followed by elimination after i.v. dosing (Figure 5A); however, PMX53 displayed a much longer half-life (1.3 h) compared with JPE-1375 (0.13 h; Table 1). We further correlated the pharmacokinetic and pharmacodynamic profiles of PMX53 and JPE-1375 and developed a pharmacokinetic/pharmacodynamic model. For PMX53, the percentage inhibition of PMN mobilization and TNF production decreased from 100 to ∼60% with a decrease in plasma PMX53 concentrations (Figure 5B). We further found a moderate negative correlation between PMN mobilization (r = 0.59) and TNF production (r = 0.57), with plasma PMX53 concentrations (Figure 5B). For JPE-1375, the percentage inhibition of PMN mobilization decreased from ∼80 to 20%, while TNF production decreased from 100 to ∼50% with a decrease in plasma JPE-1375 concentrations (Figure 5C). Unlike PMX53, JPE-1375 showed a strong negative correlation between PMN mobilization (r = 0.92) and TNF production (r = 0.75) with plasma JPE-1375 concentrations (Figure 5C). This indicates that although PMX53 has extended circulation exposure levels after i.v. administration compared to JPE-1375, this alone does not account for its prolonged in vivo pharmacodynamic activity.

Figure 5.

Figure 5

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In vivo PK/PD of PMX53 and JPE-1375. (A) Intravenous (i.v.) pharmacokinetics of PMX53 and JPE-1375 in WT mice. Complement C5aR1 antagonist PMX53 (red graph lines) and JPE-1375 (blue graph lines) concentration vs time profile in plasma following single i.v. bolus drug dose of 1 mg/kg of either antagonist in mice at time = 0. (B) PMX53 PK/PD correlations under 1 mg/kg i.v. administration, and (C) JPE-1375 PK/PD correlations under 1 mg/kg i.v. administration. Pharmacokinetic data analysis was performed using Phoenix WINNONLIN software using non-compartmental analysis (Certara, L.P, St. Louis, Missouri, USA, v8.1). Data points represent mean ± SEM of n = 5 mice at each time point.

Table 1. Pharmacokinetic Parameters of PMX53 and JPE-1375a.

Parameter JPE-1375 PMX53
Lambda_z (1/h) 5.23 0.53
t1/2 (h) 0.13 1.30
Cmax (μg/mL) 7.18 7.57
AUC 0–t (μg/mL·h) 2.40 2.67
AUC 0–inf_obs (μg/mL·h) 2.41 2.73
AUC 0–t/0–inf_obs 1.00 0.98
AUMC 0–inf_obs (μg/mL·h2) 0.13 2.02
MRT 0–inf_obs (h) 0.05 0.74
Vz_obs [(μg)/(μg/mL)] 2.38 20.60
Cl_obs [(μg)/(μg/mL)/h] 12.47 10.97
Vss_obs [(μg)/(μg/mL)] 0.66 8.11
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a

Pharmacokinetic parameters of PMX53 and JPE-1375 after single intravenous administration of 1 mg/kg of complement C5aR1 antagonist to WT mice. Values represent calculated parameter values using the mean of n = 5 concentration values. Cmax, max concentration of antagonist; t1/2, elimination half-life; AUC 0–t, area under the concentration–time curve from time zero to time t, i.e., 90 min; AUC 0–inf, area under the concentration–time curve from time zero to infinity; AUMC 0–inf, the total area under the first moment curve MRT, mean residence time; Vz, the volume of distribution at terminal phase; Cl, clearance; and Vss, the volume of distribution at the steady state.

Discussion

Numerous C5aR1 antagonists have been developed for the treatment of autoimmune and inflammatory disorders.5,21,33,34 However, there is no definitive published method for determining and comparing the working dose and therapeutic efficacy duration of these drugs in vivo. Knowledge of the pharmacodynamic profile of drug candidates, combined with their pharmacokinetic properties is essential to determine the optimal therapeutic dose and dosing frequency for preclinical translational studies. Hence, rapid and consistent quantitative output assays to determine the efficacy of therapeutic drugs need to be developed. The present study, therefore, validated PMN mobilization and TNF cytokine production in response to intravenously administered C5a to mice, as a method to quantify and evaluate the pharmacodynamics of C5aR1 antagonists in vivo.

The complement activation product C5a is considered one of the most potent inflammatory peptides, with a broad range of functions including chemotaxis of myeloid cells such as neutrophils, and the production of acute-phase proinflammatory cytokines.28,29,35,36 The current study validates this prior knowledge by demonstrating a large increase in PMN mobilization to the blood, and significant TNF production 60 min following i.v. injection of recombinant mouse C5a to C57BL6/J mice. The mobilized PMNs were identified as predominantly neutrophils, which aligns with these cells being the major mobilized population from bone-marrow reserves in the acute setting.29,37,38 Our results are also consistent with prior studies, where C5a was shown to induce rapid blood neutrophilia when injected i.v. in rabbits and to regulate the expression of TNF in vivo.37−41 Moreover, as C5a production in tissues also causes local accumulation of PMNs, the same method could be applied to examine C5aR1 antagonist activity in tissues. For example, intratracheal administration of C5a triggers alveolar inflammation and recruitment of PMNs to the alveolar space,42 and it would be interesting to evaluate C5a antagonist tissue pharmacodynamics using this model, especially given C5aR1’s proposed role in COVID-19 ARDS.43,44 In addition, the same method could theoretically be applied to human C5aR1-knockin mice,45 which would be advantageous for translational studies of human C5aR1 inhibitors.

C5a binds equally to two receptors, C5aR1 and C5aR2, both of which could contribute to the observed in vivo bioactivity of C5a. By using C5aR1-deficient mice, we demonstrated that ∼60% of the PMN mobilization response, and 90% of the TNF response to i.v. C5a in mice, is due to C5aR1. This aligns with recent reports of a C5a-C5aR2-signaling axis in mediating partial responses to C5a on neutrophils in vivo.19,27 Thus, the PMN mobilization component of this assay could theoretically be utilized to test the bioactivity of any future developed C5aR2 inhibitors, which may have selected therapeutic indications.21,34 C5a is also known to induce C5aR1–C5aR2 heterodimerization, which can modulate C5a-signaling.46 However, the antagonists (PMX53 and JPE-1375) applied in the current study are reported to be selective for C5aR1,21 the potential activity of these peptides on C5aR1–C5aR2 heterodimerization and C5aR2 activity in vivo could be explored in future studies.46 Interestingly, we also documented a small increase in PMN mobilization, but not TNF, in response to the i.v. injection of sterile saline alone, which may be a result of haemodynamic alterations or model-induced stress.47−49 Thus, it will be important to utilize a vehicle control administration group (for both drugs, and C5a) in future application of this method.

Using this pharmacodynamic method, we evaluated the working dose of the C5aR1 inhibitors, PMX53 and JPE-1375, in vivo. We demonstrated that both compounds effectively decreased PMN mobilization and TNF production at 1 and 3 mg/kg doses, while showing no significant effect at the 0.3 mg/kg dose. This is supported by prior studies demonstrating that PMX53 and JPE-1375 inhibited neutrophil influx at 1 mg/kg in a mouse RPAR model, an immune complex-mediated disease, which is driven by activation of the complement.11

In addition to dose determination, we also investigated the period of active inhibition for PMX53 and JPE-1375 using PMN mobilization and TNF production assays. We demonstrated that the inhibitory effect on C5a-mediated PMN mobilization and TNF production for PMX53 lasted at least 6 h, while the maximal inhibitory effect on PMN mobilization and TNF production for JPE-1375 lasted for only 0.25 h. Thus, although PMX53 and JPE-1375 had similar working doses in vivo, there was a significant difference in the effective inhibition period, highlighting the utility of this model to separate out therapeutic activities of C5aR1 inhibitors in vivo. This temporal difference in activity could be due to the nature of the peptides, where PMX53 as a cyclic peptide would be considerably more stable than the linear peptide JPE-1375 in the bloodstream and thus remain in circulation for longer. To test this, we further performed a pharmacokinetic analysis of both drugs at the same effective dose as used in the PMN/TNF assay and investigated the pharmacokinetics/pharmacodynamics relationship for both PMX53 and JPE-1375. Interestingly, although PMX53 did indeed have a longer plasma half-life compared with JPE-1375, it displayed weak correlation between its pharmacokinetic profile and pharmacodynamic profile. JPE-1375, on the other hand, demonstrated a very strong correlation between its pharmacokinetic and pharmacodynamic profiles in PMN mobilization, and a moderate correlation for TNF production. These differences in the pharmacokinetic/pharmacodynamic relationships for PMX53 and JPE-1375 could be due to the receptor-binding kinetics. PMX53 is referred to as a “pseudo-irreversible” antagonist that has a prolonged receptor residence time (i.e., slow off-rate),21,32,50 allowing sustained binding and inhibition of the receptor even after clearance of the drug. This is likely why the pharmacokinetics of these compound class will not necessarily match the pharmacodynamic profile. This is further supported by our prior study, where we demonstrated that PMX53 still had 60% of inhibition after 24 h post wash-off of PMX53 against C5a-induced ERK signaling in human monocyte-derived macrophages.21 Seow et al. also tested the receptor residence time of PMX53 in an intracellular calcium release assay, and the results indicated that the PMX53 maintains the potency in cells against C5a with a half-life of around 20 h.32 This study highlighted the dominant role of the binding kinetics, especially the resident time, of a compound on its target receptor, rather than a paradigm, where the pharmacodynamics must be directly correlated with circulating drug levels. In addition to the prolonged residence time (i.e., a slow off-rate), PMX53 also has a very high affinity and on-rate for C5aR121,32,51 and could be the reasons why it maintains a long period of efficacy despite the relatively short plasma half-life.

In conclusion, our study developed a rapid and consistent C5a-mediated pharmacodynamic model in mice. We utilized this model to effectively compare the pharmacodynamics of two C5aR1 antagonists PMX53 and JPE-1375 by measuring the inhibition of C5aR1-induced PMN mobilization and plasma TNF elevation. Using these assays, we found both PMX53 and JPE-1375 had similar minimally effective working doses of 1 mg/kg following i.v. injection. Pharmacokinetic/pharmacodynamic modeling demonstrated high correlation between blood concentrations and therapeutic activity for JPE-1375. However, the cyclic peptide, PMX53, had an extended in vivo efficacy period, which did not correlate with its pharmacokinetic profile. Thus, determination of the pharmacodynamics of C5aR1 antagonists using this method could be an effective strategy to identify lead compounds and assist in future clinical translational strategies.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00227.

  • Optimized MS parameters for pharmacokinetic analysis of PMX53 and JPE-1375 (PDF)

Author Contributions

T.M.W., J.D.L., V.K., and C.S.C. contributed to the study conception and design. Material preparation, data collection, and analysis were performed by C.S.C., V.K., D.M.G., and J.D.L. C.S.C. and J.D.L. performed the pharmacodynamic study. Pharmacokinetics study was performed by C.S.C. and V.K. D.M.G. and R.J.C. synthesized peptide compounds. C.S.C., J.D.L., and T.W.M. wrote the manuscript. All authors contributed to revisions of the manuscript.

This work was supported by the National Health and Medical Research Council of Australia (NHMRC) [Grant APP1118881].

The authors declare the following competing financial interest(s): TMW and RJC are inventors on patents pertaining to complement inhibitors for inflammatory disease. TMW has previously consulted to Alsonex Pty Ltd, and has received honorarium from Alexion Pharmaceuticals for participation in industry conferences and meetings. He holds no financial interests in either company. Other authors declare that they have no conflict of interest.

Supplementary Material

pt1c00227_si_001.pdf (37.4KB, pdf)

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