The anti-inflammatory effects of Fuzapladib in an endotoxemic porcine model

Abstract

Endotoxemia is a systemic inflammatory condition caused by lipopolysaccharide (LPS) stimulation, which produces inflammatory cytokines. Fuzapladib (FZP) inhibits the activation of adhesion molecules found on the surface of inflammatory cells, mitigating inflammation. In this study, we evaluated the therapeutic effects of fuzapladib on inflammatory cytokines and cardio-respiratory function using an LPS-induced endotoxemic porcine model. Fifteen pigs were separated into three groups: low-FZP (n=5), high-FZP (n=5), and control (n=5). Pigs were administered LPS under general anesthesia, and complete blood cell count, blood biochemistry, inflammatory cytokines, and cardio-respiratory function were evaluated. Statistical analysis was performed using a linear mixed-effects model and the Steel-Dwass test, with a significance threshold of P<0.05. During the 4 hr experimental period, one pig in the control group and two pigs in the low-FZP group died due to hypoxemia and hypotension. In the early acute changes following LPS administration, the high-FZP group maintained significantly higher arterial oxygen partial pressure and normal blood pressure compared to the control group. Although interleukin-6 levels increased in all groups during the experiment, they were significantly lower in the high-FZP group compared to the control group. Other parameters showed no clinically significant differences. In conclusion, while high-dose fuzapladib did not reduce organ damage in the porcine endotoxemia model, it suppressed interleukin-6 production, delayed the progression of deterioration, and contributed to a reduction in mortality during the observation period.

Lipopolysaccharides (LPS) produced by gram-negative bacteria induce endotoxemia and exacerbate sepsis. LPS activates leukocytes and endothelial cells, leading to the production of inflammatory cytokines and initiating systemic inflammatory response syndrome (SIRS) [11]. Activated inflammatory cells, mainly neutrophils, accumulate in organs, producing reactive oxygen species and proteolytic enzymes that damage endothelial cells and increase vascular permeability [3, 34]. Additionally, leukocytes and platelets adhere to the endothelium, triggering the coagulation cascade and obstructing microvasculature, resulting in a sepsis-like condition [9, 30]. Sepsis is a pathological condition caused by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) identified by pattern recognition receptors (PRRs), which cause the production of inflammatory cytokines that trigger a variety of host responses [24]. As sepsis progresses, several factors including reduced lung compliance, ventilation-perfusion mismatch, and pulmonary vascular endothelial cell malfunction, can lead to the onset of acute respiratory distress syndrome (ARDS) [15]. Once the inflammatory cascade of ARDS is initiated, controlling it becomes difficult, frequently resulting in lung and other vital organ malfunction and death [20, 29]. Therefore, the treatment of ARDS involves the inhibition of inflammatory mediators. This involves the use of medicines such as neutrophil elastase inhibitors and proteolytic enzyme inhibitors, which prevent the production of reactive oxygen species and proteolytic enzymes via inflammatory cell migration, adhesion, and infiltration [3, 34].

Fuzapladib, a novel anti-inflammatory drug, was licensed for pancreatitis in Japan in 2018 and obtained conditional approval from the U.S. Food and Drug Administration in 2022, making it the first drug to manage clinical symptoms under the same conditions in the United States. Its method inhibits inflammatory cell adherence to vascular endothelial cells and tissue infiltration, suppressing inflammatory cytokine production [33]. In a multicenter randomized controlled trial using dogs with clinical signs of acute pancreatitis, fuzapladib improved the clinical activity scores [25]. Lymphocyte function-associated antigen-1 (LFA-1) is found on the surface of inflammatory cells and primarily regulates cell adhesion through interactions with intercellular adhesion molecule-1 (ICAM-1). When cytokines are stimulated, an intracellular signaling cascade phosphorylates LFA-1, causing it to activate (structural change) on the surface of inflammatory cells and bind with ICAM-1 on endothelial cells [13]. Fuzapladib inhibits the activation of LFA-1 [23]. A pharmaceutical company conducted in vitro evaluation experiments using genetically engineered mouse pre-B cells expressing LFA-1 and, stimulated with stromal cell-derived factor-1 (SDF-1), demonstrated that fuzapladib at 1 μmol/L suppressed LFA-1 activity. Therefore, as a result of its anti-inflammatory effect, fuzapladib may be able to reduce the progression of ARDS by intervening early in the inflammatory cascade and inhibiting inflammatory cytokine production.

However, to our knowledge, there are few publications on the in vivo anti-inflammatory effects of fuzapladib. To investigate these effects, we used a porcine model of endotoxemia produced experimentally using Escherichia coli-derived LPS. Endo et al. found that administering LPS at 40 µg/kg over 30 min resulted in minimal changes in respiratory-circulatory parameters [6]. However, pigs given 160 µg/kg/hr LPS for 75 min developed ARDS and most died within 4 hr [5]. Therefore, this study evaluated the preventive effects of fuzapladib on organ damage and inflammatory cytokine suppression under conditions resembling ARDS and sepsis, using an intermediate dose of LPS based on these findings.

MATERIALS AND METHODS

Experimental animals

In this study, 15 clinically healthy female pigs (Landrace × Large White × Duroc, approximately 3 months old, weighing 29.0–35.3 kg) were assigned to three groups: control (n=5), low-dose fuzapladib (low-FZP; n=5), and high-dose fuzapladib (high-FZP; n=5). The study began with the control and low-FZP groups, before proceeding to the high-FZP group for reasons that will be explained later. Test pigs were obtained from a local pig farm (Naganuma-cho, Hokkaido, Japan), contracted by an experimental animal supplier (Sankyo Labo Service Corp., Inc., Tokyo, Japan), and delivered on the day of the experiment. Food was withheld from the pigs for 12 hr before each drug treatment, although they had free access to water. The Animal Care and Use Committee at Rakuno Gakuen University approved the study design (approval no. VH19B19).

Experimental preparations

1) Anesthesia method and experimental preparation: Medetomidine 40 μg/kg (Domitor Injection; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam 0.2 mg/kg (Midazolam Injection 10 mg; Sandoz K.K., Tokyo, Japan), and butorphanol 0.2 mg/kg (Vetorphale 5 mg; Meiji Animal Health Co., Ltd., Kumamoto, Japan) were administered intramuscularly (IM) into the neck longissimus muscle of the test pigs to induce sedation. Following anesthesia, a 22-gauge catheter (Surflow Indwelling Catheter 1 1/4”; Terumo Corp., Tokyo, Japan) was placed bilaterally in the auricular veins. Propofol (Propofol 28; Zoetis Japan Inc., Tokyo, Japan) was then administered intravenously via the 22-gauge catheter in the right auricular vein until tracheal intubation was achieved. Endotracheal intubation was performed using a cuffed endotracheal tube (internal diameter 7.0 mm; Cuffed Endotracheal Tube, Smiths Medical Japan Ltd., Tokyo, Japan). Following tracheal intubation, the test pigs were positioned in a supine position and oxygen sevoflurane inhalation anesthesia (OS anesthesia) was administered. In OS anesthesia, a dedicated sevoflurane vaporizer (Sevoflurane ASV-5; Kimura Medical Instruments Co., Ltd., Tokyo, Japan) was used as an external circuit vaporizer in conjunction with an inhalation anesthesia machine (Siesta 21; Kimura Medical Instruments Co., Ltd.) which administered oxygen at 2 L/min and sevoflurane (Sevoflo; Zoetis Japan Co., Ltd.) at a dial setting of 2% sevoflurane from the sevoflurane vaporizer, while adjusting the anesthetic depth to maintain the observed mean arterial pressure (MAP) at 80–90 mmHg.

A mixture of Lactated Ringer’s solution 500 mL (Solulact; Terumo Corp.), 5% glucose solution 500 mL (Terumo Glucose Injection 5%; Terumo Corp.), and a combination injection of thiamine hydrochloride, pyridoxine hydrochloride, and cyanocobalamin 2 mL (Daivitamix Injection; Harasawa Pharmaceutical Co., Ltd., Tokyo, Japan) was prepared and infused through the left auricular vein catheter using a connecting tube. This mixture was administered intravenously at a rate of 5 mL/kg/hr using a Top Animal Infusion Pump (Top Corp., Tokyo, Japan). Additionally, vecuronium (Vecuronium “F”; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) was delivered intravenously as a 0.1 mg/kg bolus injection. Following that, vecuronium was given as a constant rate infusion (CRI) at a rate of 0.6 to 1.0 mg/kg/hr via the same infusion line using a syringe pump (Top Animal Syringe Pump; Top Corp.), resulting in sufficient muscle relaxation to induce loss of spontaneous respiration. A ventilator was also used for intermittent positive pressure ventilation (IPPV) (Nuffield Anesthesia Ventilation Series 200; Penlon Ltd., Abingdon, UK). The IPPV parameters were set to a ventilation rate of 8–16 breaths/min, an inspiration:expiration ratio of 1:2, and a tidal volume adjusted to maintain the arterial carbon dioxide partial pressure (PaCO₂) between 35–45 mmHg.

After general anesthesia had been stabilized, the right neck and left inner thigh were shaved and disinfected before inserting a 16-gauge central venous catheter (Vascular Indwelling Catheter Kit; Medikit Co., Ltd., Tokyo, Japan) into the right external jugular vein using a cutdown technique. A temperature sensor housing (PV4046; Pulsion Medical Systems SE, Feldkirchen, Germany) and a pressure transducer (PiCCO Monitoring Kit PV8215; Pulsion Medical Systems) were connected to the central venous catheter, as well as a PiCCO system (PiCCO2; Pulsion Medical Systems). The catheter tip was positioned within the thoracic cavity and anterior vena cava to monitor the parameters. Using an ultrasound diagnostic device (UF-760AG PaoLus+; Fukuda Denshi Co., Ltd., Tokyo, Japan), a PiCCO catheter (PV2014L16-A; Pulsion Medical System) was inserted into the left femoral artery using the Seldinger technique and attached to the pressure transducer. The pressure transducers connected to the central venous and PiCCO catheters were zeroed at atmospheric pressure, at a height equivalent to the right atrium of each test pig.

2) Fuzapladib sodium administration experiment in endotoxemia porcine model: Following a 15 min stabilization period, arterial blood (3.5 mL) was withdrawn from the PiCCO catheter in the left femoral artery. This blood sample was then divided into 1 mL portions for complete blood cell count, blood chemistry analysis, and inflammatory cytokine measurement, with an additional volume (0.5 mL) used for blood gas analysis. Subsequently, fuzapladib sodium (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan) was administered through the 22-gauge catheter in the right auricular vein at 0.048 mg/kg IV, followed by CRI at a rate of 0.075 mg/kg/hr in the low-FZP group and 1.34 mg/kg IV, followed by a rate of 2.11 mg/kg/hr CRI in the high-FZP group. Fuzapladib sodium was diluted as needed with distilled water for injection (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) to achieve 1.9 mL IV in the low- and high-FZP groups, followed by a CRI of 3 mL/hr. In the control group, an equivalent volume of normal saline (Otsuka Pharmaceutical Co., Ltd.) was given at the same dose.

Five min after starting fuzapladib or saline administration, LPS 80 μg/kg (Escherichia coli O55:B5; Sigma, St. Louis, MO, USA) was administered via the 22-gauge catheter in the left auricular vein at a rate of 2 mL/kg/hr in normal saline, and adjusted to complete within 30 min. Complete blood cell count was performed at the start of LPS administration (T0), as well as at 15 (T15), 30 (T30), 45 (T45), 60 (T60), 90 (T90), 120 (T120), 150 (T150), 180 (T180), 210 (T210), and 240 min (T240) later. Blood chemistry analyses were performed at periods T0, T60, T120, T180, and T240. In addition, 4 mL of arterial blood was taken from the PiCCO catheter after fuzapladib administration before T0, T15, T30, T60, T120, T180, and T240 in two low-FZP and five high-FZP pigs, respectively. Using heparin as an anticoagulant, the collected blood was centrifuged at 1,000 g for 2 min in a tabletop centrifuge (ESK12500; AS ONE Corp., Osaka, Japan), and the plasma was separated and stored at −20°C until fuzapladib concentrations were measured. At the end of the experiment, pentobarbital sodium (30 mg/kg; Somnopentyl; Kyoritsu Seiyaku Pharmaceuticals, Tokyo, Japan) was rapidly injected intravenously to euthanize all pigs under OS anesthesia. This was followed by the intravenous injection of potassium chloride (20 mEq/head; KCL Correction Solution 1 mEq/mL; Otsuka Pharmaceutical Co., Ltd.) to guarantee euthanasia.

Evaluation for cardio-respiratory function

Vital signs, including heart rate (HR: beats/min), respiratory rate (RR: breaths/min), body temperature (Temp: °C), and end-tidal sevoflurane concentration (ETSEV: %), were continuously measured and recorded during OS anesthesia using an animal vital sign monitor (BP-608; Omron Colin Co., Ltd., Tokyo, Japan). In addition, a pulse oximeter probe (RE Rainbow Lite SET Neo; Masimo Japan Corp., Tokyo, Japan) was attached to the shaved tail to measure and record the transcutaneous arterial oxygen saturation (SpO₂, %) and perfusion index (PI, %), while the pleth variability index (PVI, %) was measured using a pulse CO-oximeter (New Radical-7; Masimo Japan Corp.).

Following a 15 min stabilization phase, 10 mL of 0°C normal saline was delivered through the central venous catheter at all time points, and temperature changes were recorded using the PiCCO catheter. The PiCCO system measures transpulmonary cardiac output (TPTDCO, L/min) using the thermodilution curve formed by blood temperature changes (Stewart-Hamilton method), and blood volumes for each compartment (right atrium, right ventricle, lungs, left atrium, left ventricle) are calculated as saline flows through the thoracic compartments [16]. Simultaneously, central venous pressure (CVP, mmHg) was measured, and arterial blood pressures (systolic arterial pressure (SAP), mean arterial pressure (MAP, mmHg), and diastolic arterial pressure (DAP)) as well as left ventricular contractility index (dPmx, mmHg/sec) were invasively measured and recorded using a PiCCO catheter placed in the femoral artery. These values were used to calculate stroke volume (SV: mL), stroke volume variation (SVV: %), pulse pressure variation (PPV: %), systemic vascular resistance (SVR: dyne·sec·cm^-5), global end-diastolic volume (GEDV: mL), extravascular lung water (EVLW: mL), global ejection fraction (GEF:%), and pulmonary vascular permeability index (PVPI) (Supplementary File 1) [16].

Measurement for blood gas, complete blood cell count, blood chemistry, and inflammatory cytokine

Blood gases were analyzed at all time points using a blood gas analyzer (GEM3500; IL Japan Co., Ltd., Tokyo, Japan). A 0.5 mL subsample of the 3.5 mL arterial blood sample collected via the PiCCO catheter was treated with 60 units of sodium heparin (Novo-Heparin 1,000 units/mL; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan). The subsample was analyzed for arterial blood pH (pHa), partial pressure of arterial oxygen (PaO₂: mmHg), PaCO₂ (mmHg), blood lactate (mmol/L), bicarbonate ion (HCO₃⁻: mEq/L), base excess (B.E.: mmol/L), sodium ion (Na⁺: mEq/L), potassium ion (K⁺: mEq/L), and ionized calcium (iCa: mmol/L).

A 1 mL subsample of the 3.5 mL samples collected at all time points was aliquoted into EDTA tubes (EDTA-2K; Sekisui Medical Co., Ltd., Tokyo, Japan), inverted to mix, and analyzed using a multi-parameter automated hematology analyzer (pocH-100iV; Sysmex Corporation, Kobe, Japan) to measure white blood cell count (WBC: /μL), red blood cell count (RBC: 10^6/μL), hemoglobin (Hb: mg/dL), hematocrit (Ht: %), and platelet count (PLT: 10^4/μL).

Simultaneously, 1 mL of the 3.5 mL collected at T0, T60, T120, T180, and T240 was aliquoted into heparin tubes (Fujifilm Heparin Tube; Fujifilm Corporation, Tokyo, Japan), centrifuged at 1,000 g for 2 min using a benchtop centrifuge, and frozen at −20°C. A biochemical autoanalyzer (BioMajesty JCA-BM6010 G; JEOL Ltd., Tokyo, Japan) was used to measure albumin (ALB: g/dL), glucose (Glu: mg/dL), blood urea nitrogen (BUN: mg/dL), creatinine (CREA: mg/dL), aspartate aminotransferase (AST: IU/L), alanine aminotransferase (ALT: IU/L), alkaline phosphatase (ALP: IU/L), γ-glutamyl transferase (γ-GTP: IU/L), total bilirubin (T-Bil: mg/dL), total bile acids (TBA: mmol/L), triglycerides (TG: mg/dL), total cholesterol (T-Cho: mg/dL), and lipase (Lip: IU/L) concentrations in the thawed plasma.

In addition, 1 mL of the 3.5 mL collected at all time points was aliquoted into heparin tubes (Fujifilm Heparin Tube; Fujifilm), separated to extract plasma, and frozen at −20°C. Inflammatory cytokines including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-α, and IFN-γ were measured using the sandwich ELISA method (Cytokine & Chemokine 9-Plex Porcine ProcartaPlex Panel 1; Thermo Fisher Scientific Inc., Tokyo, Japan).

Measurement of fuzapladib concentrations in blood

The dosage for the low-FZP group was calculated based on the pharmacokinetic parameters of fuzapladib obtained from the drug company’s disclosure and the effective concentration determined from the aforementioned in vitro evaluation test using LFA-1 introduced in genetically modified mouse pre-B cells (1 µmol/L=0.4294 µg/mL). However, because the low-FZP group showed no clinically meaningful effects, we determined fuzapladib plasma concentration in the last two pigs. Based on the pharmacokinetic data acquired from the low-FZP group, the dosage for the high-FZP group was established at ten times the initial target plasma concentration (10 µmol/L=4.294 µg/mL). Blood samples were collected from all pigs in the high-FZP group. The blood samples from the two pigs in the low-FZP group and all pigs in the high-FZP group were sent to Ishihara Sangyo Kaisha Ltd., the company that developed fuzapladib, to determine the blood fuzapladib levels using high-performance liquid chromatography.

Statistical analysis

Excel statistics (BellCurve for Excel; Social Survey Research Information Co., Ltd., Tokyo, Japan) were used to analyze the sample size and the intergroup differences. Based on the report of circulatory changes in an endotoxemia porcine model study by Endo et al. [6] (control group: MAP=90 ± 10 mmHg vs. LPS group: MAP=50 ± 10 mmHg), the sample size was calculated using the Hedges’ g effect size of 1.00, assuming a specific effect (70 ± 10 mmHg) with fuzapladib administration. Using α=0.05 and β=0.8, the sample size was calculated as 4.9, resulting in five pigs in each group. Kruskal-Wallis and Steel-Dwass tests were used to assess the intergroup differences in each parameter at each time point following LPS administration. The Steel test evaluated intragroup differences in each parameter over time following LPS administration. The statistical program EZR (Easy R version 1.61; Jichi Medical University, Shimotsuke, Japan) was used to analyze the interaction between time points and groups for each parameter following LPS administration using a linear mixed-effects model. Statistical significance was defined as P<0.05 in all analyses.

RESULTS

Outcome of endotoxemia porcine model

One of the control pigs died 130 min after receiving LPS due to severe hypotension and hypoxemia. Two pigs in the low-FZP group died 157 and 222 min after receiving LPS, respectively, due to severe hypotension and hypoxemia. In contrast, all pigs in the high-FZP group survived the whole experimental period and were euthanized.

Changes in cardio-respiratory function

1) Changes in cardiovascular function: Table 1 shows the trends in cardiovascular parameters in the control, low-FZP, and high-FZP groups. Following LPS administration, all groups exhibited significant decreases in TPTDCO (P<0.001), SV (P<0.001), dPmx (P=0.013), GEF (P<0.001), SAP (P<0.001), MAP (P<0.001), DAP (P<0.001), GEDV (P<0.001), and PI (P<0.001). TPTDCO, SV, MAP, and GEDV showed significant declines. During the observation period, severe hypotension (MAP <50 mmHg) was reported in three pigs in the control group, five pigs in the low-FZP group, and four pigs in the high-FZP group, with no significant difference in the incidence rate (P=0.2865). In contrast, all groups experienced significant increases in Temp (P=0.009), HR (P<0.001), SVV (P=0.021), and PPV (P=0.030) over time, particularly in HR. Although SVR did not show significant changes over time, the high-FZP group was significantly higher than the control group at T60 and T90 (P=0.0087 and P=0.0263, respectively), and MAP was significantly lower in the control group at T60 than the high-FZP group (P=0.0184).

Table 1. Changes in cardiovascular parameters over time following lipopolysaccharide (LPS) administration.

		Elapsed time after LPS administration (min)								FZP:Time
		0	15	30	60	90	120	180	240	P-value
HR	Control#	109 (5)	101 (5)	101 (5)	112 (5)	134 (5)	154 (5)	178 (4)	198 (4)
(beats/min)		[101–112]	[93–104]	[84–104]	[103–127]	[114–154]	[110–180]	[156–183]	[171–213]
	Low-FZP#	107 (5)	98 (5)	101 (5)	108 (5)	128 (5)	147 (5)	161 (4)	165 (3)	0.87
		[93–111]	[85–109]	[82–118]	[91–138]	[98–147]	[119–168]	[151–173]	[131–176]
	High-FZP#	99 (5)	99 (5)	97 (5)	103 (5)	122 (5)	144 (5)	164 (5)	177 (5)	0.91
		[96–106]	[92–107]	[94–101]	[91–110]	[90–126]	[98–154]	[118–178]	[162–181]
MAP	Control♭	77 (5)	98 (5)	97 (5)	58 (5)	52 (5)	53 (4)	51 (4)	54 (4)
(mmHg)		[75–93]	[82–105]	[72–105]	[39–60]	[33–57]	[25–59]	[49–52]	[50–58]
	Low-FZP♭	77 (5)	86 (5)	90 (5)	55 (5)	48 (5)	48 (5)	42 (4)	40 (3)	0.35
		[72–83]	[82–101]	[83–97]	[49–60]	[37–54]	[37–54]	[39–48]	[34–48]
	High-FZP♭	92 (5)	101 (5)	104 (5)	67 (5)*	64 (5)	55 (5)	50 (5)	50 (5)	0.16
		[72–93]	[94–121]	[96–116]	[59–72]	[49–72]	[44–63]	[40–58]	[43–61]
TPTDCO	Control♭	3.85 (5)	2.46 (5)	2.82 (5)	3.20 (5)	2.97 (5)	2.45 (5)	2.09 (4)	1.50 (3)
(L/min)		[3.01–4.34]	[2.08–2.77]	[1.93–3.75]	[2.62–3.88]	[2.24–3.27]	[2.24–3.27]	[1.57–2.12]	[1.28–2.07]
	Low-FZP♭	3.78 (5)	2.13 (5)	2.72 (5)	2.83 (5)	2.71 (5)	2.53 (5)	2.15 (4)	1.50 (3)	0.39
		[3.45–4.71]	[1.25–2.86]	[2.20–2.90]	[2.64–4.11]	[2.26–3.34]	[1.95–2.90]	[1.84–2.35]	[1.28–2.07]
	High-FZP♭	3.35 (5)	2.61 (5)	2.62 (5)	2.97 (5)	2.68 (5)	2.39 (5)	1.97 (5)	1.82 (5)	0.33
		[2.93–3.98]	[2.32–3.23]	[2.02–3.17]	[2.78–3.26]	[2.19–3.17]	[1.92–2.73]	[1.61–2.13]	[1.53–2.17]
SV	Control♭	34.5 (5)	23.9 (5)	26.6 (5)	27.7 (5)	21.3 (5)	14.3 (5)	11.1 (4)	9.0 (4)
(mL/beat)		[29.3–41.3]	[21.3–29.7]	[20.0–30.3]	[24.5–32.0]	[18.8–24.3]	[11.5–17.3]	[10.0–11.7]	[6.0–12.0]
	Low-FZP♭	40.0 (5)	24.3 (5)	27.5 (5)	28.0 (5)	22.7 (5)	16.3 (5)	13.2 (4)	10.5 (3)	0.77
		[36.7–46.0]	[12.3–27.3]	[21.3–41.7]	[25.0–36.7]	[19.3–23.0]	[15.0–21.0]	[11.7–15.0]	[9.0–12.8]
	High-FZP♭	32.3 (5)	25.3 (5)	25.3 (5)	29.3 (5)	22.0 (5)	17.5 (5)	12.0 (5)	10.5 (5)	0.95
		[30.5–39.0]	[23.3–33.0]	[23.3–33.0]	[27.0–32.6]	[18.0–29.7]	[12.0–24.5]	[10.0–17.0]	[8.3–13.0]
dPmx	Control♭	521 (5)	419 (5)	592 (5)	580 (5)	349 (5)	328 (5)	328 (4)	394 (3)
(mmHg/sec)		[371–794]	[341–595]	[339–829]	[371–869]	[265–566]	[222–437]	[225–481]	[335–586]
	Low-FZP♭	697 (5)	519 (5)	796 (5)	723 (5)	487 (5)	372 (5)	287 (4)	253 (3)	0.08
		[593–864]	[491–748]	[549–986]	[566–984]	[347–509]	[242–438]	[217–642]	[242–343]
	High-FZP♭	746 (5)	673 (5)	758 (5)	774 (5)	458 (5)	347 (5)	353 (5)	439 (4)	0.55
		[437–787]	[457–763]	[461–841]	[416–1,114]	[366–1,048]	[287–736]	[269–581]	[298–717]
SVR	Control	1,403 (5)	2,878 (5)	2,660 (5)	1,035 (5)	1,215 (5)	1,443 (5)	1,641 (4)	2,340 (4)
(dyne·sec·cm-5)		[1,295–2,240]	[2,363–3,217]	[1,298–4,032]	[723–1,528]	[730–1,430]	[700–1,963]	[1,510–2,086]	[1,597–3,083]
	Low-FZP	1,356 (5)	2,816 (5)	2,430 (5)	1,243 (5)$	1,093 (5)	1,163 (5)	1,222 (4)	1,582 (3)	0.66
		[1,050–1,660]	[2,386–3,923]	[1,870–2,980]	[980–1,503]	[1,043–1,766]	[957–2,020]	[1,157–1,653]	[1,268–2,227]
	High-FZP	1,818 (5)	3,068 (5)	3,414 (5)	1,722 (5)*	1,633 (5)*	1,780 (5)	1,728 (5)	1,973 (5)	0.29
		[1,778–2,157]	[2,210–3,527]	[2,210–3,527]	[1,560–1,770]	[1,556–2,430]	[1,372–2,400]	[1,493–2,645]	[1,902–2,937]
GEDV	Control♭	401 (5)	415 (5)	439 (5)	352 (5)	303 (5)	293 (5)	299 (4)	293 (4)
(mL)		[385–607]	[376–610]	[384–622]	[321–520]	[286–509]	[270–470]	[264–471]	[197–517]
	Low-FZP♭	489 (5)	464 (5)	498 (5)	384 (5)	368 (5)	360 (5)	317 (4)	270 (3)	0.48
		[401–570]	[321–566]	[356–520]	[313–456]	[301–421]	[291–426]	[283–383]	[254–363]
	High-FZP♭	381 (5)	364 (5)	400 (5)	350 (5)	315 (5)	295 (5)	259 (5)	272 (5)	0.91
		[353–566]	[356–531]	[343–547]	[289–475]	[255–425]	[242–384]	[256–323]	[245–377]
PI	Control♭	2.75 (5)	1.63 (5)	1.78 (5)	0.77 (5)	0.22 (5)	0.45 (3)	0.15 (2)	0.37 (2)
(%)		[1.63–5.62]	[0.39–2.20]	[0.95–3.8]	[0.46–1.9]	[0–1.10]	[0.10–1.50]	[0.12–0.18]	[0.32–0.43]
	Low-FZP♭	3.80 (5)	1.90 (5)	1.40 (5)	0.78 (5)	0.98 (5)	0.38 (5)	0.16 (3)	0.27 (2)	0.59
		[2.00–7.20]	[1.04–2.50]	[1.22–2.38]	[0.31–4.65]	[0.09–2.63]	[0.22–0.94]	[0.07–0.46]	[0.21–0.33]
	High-FZP♭	2.02 (5)	0.85 (5)	0.87 (5)	1.67 (5)	1.53 (5)	0.83 (5)	0.25 (4)	0.13 (4)	0.25
		[0.63–4.98]	[0.65–1.88]	[0.49–1.73]	[1.07–3.92]	[0.70–1.60]	[0.32–1.25]	[0.18–0.68]	[0.12–0.29]

Values represent the median (number of pigs) [min-max]. Fuzapladib (FZP): time: P-values indicate the P-values for the interaction between fuzapladib dosage and elapsed time after LPS in a linear mixed-effects model. * vs. control group (P<0.05); $ vs. High-FZP group (P<0.05). #: There was a significant increase in temporal changes within each group (P<0.05). ♭: There was a significant decrease in temporal changes within each group (P<0.05). HR: heart rate, MAP: mean arterial blood pressure, TPTDCO: cardiac output measured using transpulmonary thermodilution, SV: stroke volume, dPmx: left ventricular contractility index, SVR: systemic vascular resistance, GEDV: global end-diastolic volume, PI: perfusion index. For other complete blood cell count and biochemical test results, please refer to the Supplementary File 2.

2) Changes in respiratory function and acid-base balance: Table 2 shows the trends in respiratory function and acid-base balance parameters in the control, low-FZP, and high-FZP groups. Following LPS administration, all groups saw significant decreases in PaO₂ (P=0.002), pHa (P<0.001), B.E. (P<0.001), and HCO₃⁻ (P<0.001). The number of pigs with PaO₂ <200 mmHg during 100% oxygen inhalation was three in the control group, four in the low-FZP group, and one in the high-FZP group (P=0.1534). Across all groups, lactate (P=0.001) and PVPI (P<0.01) levels increased significantly over time. There was no significant difference in EVLW across the groups; however, one of the pigs that exceeded 14 mL/kg was in the control group, two in the low-FZP group, and one in the high-FZP group. Three pigs in the control and low-FZP groups died during the study. The high-FZP group had significantly higher PaO₂ levels at T45 and T60 than the control group (P=0.0134 and P=0.0250, respectively).

Table 2. Changes in respiratory parameters and blood gas analysis over time following lipopolysaccharide (LPS) administration.

		Elapsed time after LPS administration (min)								FZP:Time
		0	15	30	45	60	120	180	240	P-value
PaO2	Control♭	458 (5)	577 (4)	507 (4)	337 (5)	157 (5)	377 (5)	248 (4)	210 (4)
(mmHg)		[314–548]	[548–585]	[220–572]	[83–438]	[65–298]	[108–439]	[122–423]	[94–373]
	Low-FZP♭	445 (5)	573 (5)	470 (5)	455 (5)	404 (5)	351 (5)	203 (4)	145 (4)	0.25
		[285–509]	[538–584]	[231–567]	[135–525]	[84–451]	[99–430]	[53–331]	[50–250]
	High-FZP♭	464 (5)	538 (5)*	523 (5)	515 (5)*	431 (5)*	424 (5)	331 (5)	302 (4)	0.88
		[41–519]	[500–546]	[430–546]	[501–525]	[255–505]	[272–460]	[117–375]	[99–354]
pHa	Control♭	7.56 (5)	7.54 (4)	7.48 (4)	7.42 (5)	7.40 (5)	7.45 (5)	7.38 (4)	7.32 (4)
		[7.49–7.57]	[7.49–7.58]	[7.43–7.56]	[7.28–7.50]	[7.23–7.46]	[7.32–7.51]	[7.19–7.46]	[7.05–7.45]
	Low-FZP♭	7.54 (5)	7.58 (5)	7.51 (5)	7.45 (5)	7.43 (5)	7.40 (5)	7.36 (4)	7.33 (4)	0.73
		[7.5–7.57]	[7.54–7.60]	[7.34–7.53]	[7.37–7.48]	[7.38–7.45]	[7.32–7.44]	[7.22–7.41]	[7.07–7.39]
	High-FZP♭	7.55 (5)	7.57 (5)	7.52 (5)	7.51 (5)*	7.47 (5)	7.43 (5)	7.40 (5)	7.34 (5)	0.59
		[7.53–7.57]	[7.54–7.58]	[7.42–7.53]	[7.49–7.52]	[7.44–7.49]	[7.37–7.50]	[7.32–7.44]	[7.28–7.41]
PaCO2	Control	40 (5)	40 (4)	38 (4)	45 (5)	47 (5)	42 (5)	42 (4)	44 (4)
(mmHg)		[36–47]	[35–47]	[33–43]	[35–51]	[40–60]	[35–57]	[32–72]	[34–82]
	Low-FZP	41 (5)	38 (5)	37 (5)	40 (5)	43 (5)	43 (5)	44 (4)	46 (4)	0.57
		[39–47]	[33–41]	[34–51]	[33–48]	[36–53]	[40–53]	[39–69]	[36–79]
	High-FZP	39 (5)	38 (5)	37 (5)	36 (5)	39 (5)*	40 (5)	40 (5)	43 (5)	0.67
		[36–42]	[34–48]	[36–44]	[35–39]	[37–41]	[36–46]	[38–46]	[36–46]
HCO3−	Control♭	35.6 (5)	34.5 (5)	27.1 (5)	28.2 (5)	28.5 (5)	27.1 (5)	24.6 (4)	22.2 (4)
(mEq/L)		[32.4–37.6]	[31.7–35.9]	[26.1–30.0]	[23.7–28.5]	[24.1–29.7]	[22.6–29.1]	[21.0–28.4]	[17.2–27.7]
	Low-FZP♭	33.6 (5)	34.5 (5)	27.1 (5)	25.4 (5)	26.0 (5)	26.6 (5)	24.3 (4)	22.2 (3)	0.63
		[33.1–37.4]	[31.6–35.3]	[23.4–30.4]	[23.7–31.3]	[25.0–31.1]	[23.7–30.4]	[23.5–27.6]	[21.2–23.6]
	High-FZP♭	33.9 (5)	33.8 (5)	29.7 (5)	28.2 (5)	27.1 (5)	26.5 (5)	24.1 (5)	22.4 (5)	0.55
		[31.9–35.5]	[31.3–34.5]	[28.4–30.8]	[28.0–29.4]	[26.8–28.9]	[25.7–29.2]	[23.2–26.3]	[19.7–24.3]
B.E.	Control♭	11.5 (5)	11.7 (5)	4.8 (5)	4.2 (5)	4.0 (5)	3.7 (5)	-1.1 (4)	-4.8 (4)
(mmol/L)		[10.1–14.1]	[8.4–13.4]	[2.9–8.1]	[3.0–4.7]	[3.1–4.1]	[2.1–5.0]	[-4.2–4.2]	[-10.1–3.8]
	Low-FZP♭	11.0 (5)	11.7 (5)	4.9 (5)	2.0 (5)	2.0 (5)	1.9 (5)	-0.8 (4)	-3.1 (3)	0.99
		[10.1–14.4]	[10.1–12.8]	[-0.7–7.9]	[0.3–6.7]	[1.2–5.8]	[-1.7–4.7]	[-1.1– -0.1]	[-8.3– -2.6]
	High-FZP♭	11.0 (5)	11.1 (5)	7.0 (5)	5.6 (5)*	4.0 (5)	3.3 (5)	-0.4 (5)	-1.3 (5)	0.55
		[9.4–12.8]	[8.6–11.9]	[4.1–7.8]	[5.5–6.3]	[3.2–5.4]	[1.0–6.3]	[-2.4–2.4]	[-6.3– -0.8]
Lactate	Control#	1.2 (5)	1.3 (5)	2.8 (5)	3.4 (5)	3.5 (5)	3.4 (5)	4.9 (4)	6.4 (4)
(mmol/L)		[1.0–1.2]	[1.0–1.6]	[2.1–4.0]	[2.1–3.9]	[2.1–3.7]	[3.3–8.5]	[3.1–8.2]	[3.0–11.4]
	Low-FZP#	1.0 (5)	1.2 (5)	3.5 (5)	3.8 (5)	3.5 (5)	3.4 (5)	5.3 (4)	6.2 (3)	0.44
		[0.8–1.2]	[0.8–1.7]	[2.7–4.5]	[3.1–3.9]	[2.8–3.9]	[3.0–5.0]	[3.6–6.4]	[3.9–10.0]
	High-FZP#	0.9 (5)	1.2 (5)	2.0 (5)	2.4 (5)	2.4 (5)	2.9 (5)	4.0 (5)	4.6 (5)	0.27
		[0.6–1.1]	[0.9–1.3]	[1.3–4.0]	[1.7–4.8]	[2.1–4.6]	[2.4–4.2]	[3.6–6.2]	[3.9–7.9]
EVLW	Control	283 (5)	274 (5)	297 (5)	337 (5)	315 (5)	291 (5)	330 (4)	359 (4)
(%)		[250–300]	[239–317]	[286–432]	[323–363]	[289–339]	[283–474]	[307–381]	[274–428]
	Low-FZP	374 (5)	392 (5)	378 (5)	373 (5)	354 (5)	360 (5)	399 (4)	407 (3)	0.82
		[292–378]	[290–478]	[254–412]	[284–414]	[261–415]	[272–461]	[307–577]	[367–412]
	High-FZP	311 (5)	309 (5)	293 (5)	345 (5)	342 (5)	312 (5)	308 (5)	352 (5)	0.51
		[282–338]	[293–339]	[284–354]	[289–406]	[285–384]	[288–368]	[287–433]	[315–466]
PVPI	Control#	2.62 (5)	2.45 (5)	2.80 (5)	3.78 (5)	4.02 (5)	3.83 (5)	4.68 (4)	4.91 (4)
(%)		[1.80–2.80]	[2.00–2.77]	[1.80–3.88]	[2.50–4.50]	[2.50–4.38]	[2.20–6.40]	[2.65–4.97]	[2.08–6.40]
	Low-FZP#	2.73 (5)	3.17 (5)	2.95 (5)	3.57 (5)	3.67 (5)	3.60 (5)	4.61 (4)	6.16 (3)	0.72
		[2.50–3.57]	[2.77–5.33]	[2.35–4.55]	[3.10–4.77]	[3.13–5.37]	[3.16–5.83]	[4.10–6.97]	[3.90–6.50]
	High-FZP#	3.03 (5)	3.15 (5)	3.03 (5)	3.88 (5)	4.30 (5)	4.30 (5)	4.73 (5)	5.20 (5)	0.64
		[2.38–3.53]	[2.40–3.37]	[2.50–3.38]	[3.27–3.93]	[3.60–4.37]	[3.37–4.80]	[4.20–5.57]	[3.68–6.73]

Values represent the median (number of pigs) [min–max]. Fuzapladib (FZP): Time: P-values indicate the P-values for the interaction between the dosage of fuzapladib and the elapsed time after LPS in a linear mixed-effects model. *: vs. control group (P<0.05), #: There was a significant increase in temporal changes within each group (P<0.05). ♭: There was a significant decrease in temporal changes within each group (P<0.05). PaO₂: partial pressure of arterial oxygen, pHa: pH of arterial blood, PaCO₂: partial pressure of arterial carbon dioxide, HCO₃⁻: bicarbonate ion, B.E.: base excess, EVLW: extravascular lung water, PVPI: pulmonary vascular permeability index. For other complete blood cell count and biochemical test results, please refer to Supplementary File 3.

Changes in complete blood cell count, blood biochemistry, inflammatory cytokine parameters, and fuzapladib concentration in blood

Table 3 highlights changes in the complete blood cell count and blood biochemical parameters. Following LPS administration, all groups experienced significant decreases in WBC (P<0.001), PLT (P<0.001), Na⁺ (P=0.002), ALB (P<0.001), ALT (P=0.011), T-Cho (P=0.004), and glucose (P=0.001) levels over time. RBC (P<0.001), hemoglobin (P<0.001), Ht (P<0.001), K⁺ (P<0.001), ALP (P<0.001), γ-GTP (P=0.046), T-Bil (P<0.001), TBA (P=0.014), BUN (P<0.001), and CREA (P<0.001) all increased significantly with time. Several parameters changed dramatically during the observation period, most notably a marked decrease in glucose across all groups, whereas CREA increased significantly across all groups. The high-FZP group showed a significant decrease in CREA compared to the low-FZP group at T60 and T120 (P<0.05). Furthermore, although clinically insignificant, T-Bil levels increased considerably in the high-FZP group compared to the low-FZP group over time. Potassium levels increased significantly over time in all groups, with values at T240 of 5.8 [5.4–6.4] mmol/L for the control group, 5.45 [4.9–8.3] mmol/L for the low-FZP group, and 5.2 [4.9–5.8] mmol/L for the high-FZP group (median [minimum-maximum]). All groups showed significant increases in ALP and γ-GTP levels over time (P<0.05).

Table 3. Changes in complete blood cell count and biochemical parameters over time following lipopolysaccharide (LPS) administration.

		Elapsed time after LPS administration (min)								FZP:Time
		0	15	30	45	60	120	180	240	P-value
WBC	Control♭	24,900 (5)	14,500 (5)	4,100 (5)	4,400 (5)	5,800 (5)	4,300 (5)	3,150 (4)	3,150 (4)
(/μL)		[17,400–30,000]	[11,200–21,500]	[3,300–10,900]	[3,700–5,700]	[4,700–9,600]	[2,900–6,400]	[1,800–3,700]	[1,900–4,300]
	Low-FZP♭	23,100 (5)	12,400 (5)	3,300 (5)	4,200 (5)	5,500 (5)	3,400 (5)	2,300 (4)	2,100 (3)	0.74
		[13,800–30,000]	[9,600–19,600]	[2,900–3,700]	[3,300–5,700]	[4,700–7,900]	[3,000–4,800]	[2,200–2,900]	[2,000–3,200]
	High-FZP♭	18,600 (5)	12,400 (5)	4,500 (5)	4,300 (5)	5,300 (5)	3,300 (5)	2,700 (5)	2,500 (5)	0.65
		[13,500–39,700]	[9,700–17,000]	[2,900–8,400]	[2,900–7,000]	[3,000–11,300]	[2,100–6,900]	[1,500–5,700]	[1,200–6,800]
Ht	Control#	27.4 (5)	30.6 (5)	37.9 (5)	36.5 (5)	33.6 (5)	36.5 (5)	40.4 (4)	42.8 (4)
(%)		[23.0–31.3]	[25.3–34.9]	[35.5–40.6]	[34.1–38.6]	[28.3–37.2]	[26.3–39.6]	[38.2–42.6]	[40.0–48.1]
	Low-FZP#	30.0 (5)	33.4 (5)	38.6 (5)	36.9 (5)	35.7 (5)	36.2 (5)	36.4 (4)	39.3 (3)	0.02
		[26.9–31.3]	[31.0–36.8]	[36.7–39.5]	[33.2–38.3]	[31.6–37.2]	[32.7–36.8]	[34.1–41.8]	[35.5–43.2]
	High-FZP#	28.9 (5)	31.8 (5)	37.0 (5)	35.6 (5)	35.0 (5)	36.6 (5)	38.2 (5)	40.3 (5)	0.23
		[25.5–31.7]	[28.2–35.2]	[32.2–38.5]	[31.6–38.7]	[31.9–38.2]	[32.1–38.8]	[33.0–41.2]	[35.5–42.7]
PLT	Control♭	54.5 (5)	53.9 (5)	47.2 (5)	44.2 (5)	41.8 (5)	32.1 (5)	25.0 (4)	18.6 (4)
(104/μL)		[49.7–76.7]	[47.4–84.3]	[34.4–62.9]	[32.3–65.2]	[34.3–66.2]	[23.8–60.7]	[20.4–31.1]	[15.8–31.2]
	Low-FZP♭	52.8 (5)	49.3 (5)	47.5 (5)	44.7 (5)	44.1 (5)	28.1 (5)	19.0 (4)	18.4 (3)	0.85
		[42.5–59.1]	[40.7–62.0]	[28.5–47.9]	[29.8–46.8]	[32.1–46.6]	[24.3–34.3]	[16.9–29.3]	[15.8–28.9]
	High-FZP♭	43.3 (5)	43.4 (5)	35.4 (5)	32.3 (5)	35.1 (5)	28.0 (5)	22.8 (5)	22.9 (5)	0.12
		[30.1–64.4]	[28.8–66.9]	[23.9–52.3]	[22.5–50.4]	[20.0–52.2]	[16.3–36.8]	[14.2–27.3]	[13.3–25.6]
ALB	Control♭	2.8 (5)				2.7 (5)	2.5 (5)	2.4 (4)	2.5 (4)
(mg/dL)		[2.4–3.3]				[2.3–3.3]	[2.1–3.1]	[2.1–2.9]	[2.0–2.6]
	Low-FZP♭	2.9 (5)				2.8 (5)	2.6 (5)	2.5 (4)	2.4 (3)	0.91
		[2.6–3.1]				[2.5–3.1]	[2.3–2.7]	[2.3–2.5]	[2.4–2.5]
	High-FZP♭	3.0 (5)				3.0 (5)	2.6 (5)	2.4 (5)	2.4 (5)	0.60
		[2.6–3.3]				[2.7–3.4]	[2.4–3.3]	[2.3–3.0]	[2.2–2.9]
ALP	Control#	192 (5)				240 (5)	267 (5)	298 (4)	358 (4)
(IU/L)		[119–221]				[139–289]	[181–344]	[200–371]	[269–402]
	Low-FZP#	172 (5)				189 (5)	197 (5)	194 (4)	217 (3)	0.16
		[100–210]				[119–282]	[148–338]	[172–338]	[206–349]
	High-FZP#	207 (5)				239 (5)	278 (5)	282 (5)	330 (5)	0.64
		[137–225]				[218–283]	[252–313]	[256–398]	[290–403]
Glucose	Control♭	145 (5)				135 (5)	110 (5)	45 (4)	35 (4)
(mg/dL)		[126–153]				[100–168]	[45–132]	[13–140]	[13–90]
	Low-FZP♭	153 (5)				143 (5)	98 (5)	50 (4)	24 (3)	0.43
		[133–176]				[126–148]	[79–114]	[26–104]	[10–141]
	High-FZP♭	131 (5)				137 (5)	97 (5)	52 (5)	20 (5)	0.58
		[100–152]				[75–153]	[20–111]	[10–90]	[10–76]
T-Bil	Control#	0.04 (5)				0.03 (5)	0.18 (5)	0.56 (4)	0.78 (4)
(mg/dL)		[0.01–0.13]				[0.01–0.12]	[0.01–0.26]	[0.23–0.67]	[0.40–0.94]
	Low-FZP#	0.03 (5)				0.03 (5)	0.11 (5)	0.29 (4)	0.52 (3)	0.12
		[0.01–0.08]				[0.01–0.09]	[0.08–0.17]	[0.27–0.45]	[0.51–0.62]
	High-FZP#	0.06 (5)				0.08 (5)*	0.14 (5)*	0.47 (5)*	0.81 (5)*	<0.01
		[0.02–0.07]				[0.02–0.09]	[0.07–0.31]	[0.32–0.66]	[0.43–0.92]
CREA	Control#	0.77 (5)				0.90 (5)	1.17 (5)	1.42 (4)	1.60 (4)
(mg/dL)		[0.63–0.83]				[0.71–1.10]	[1.01–1.37]	[1.28–1.49]	[1.58–1.73]
	Low-FZP#	0.81 (5)				0.90 (5)	1.30 (5)	1.55 (4)	1.78 (3)	0.43
		[0.74–0.83]				[0.78–1.01]	[1.12–1.32]	[1.39–1.66]	[1.67–1.87]
	High-FZP#	0.75 (5)				0.72 (5)*	0.97 (5)*	1.37 (5)	1.46 (5)	0.13
		[0.67–0.79]				[0.67–0.78]	[0.80–1.20]	[1.05–1.47]	[1.26–1.77]

Values represent the median (number of pigs) [min-max]. Fuzapladib (FZP): Time: P-values indicate the P-values for the interaction between the dosage of fuzapladib and the elapsed time after LPS in a linear mixed-effects model. *: vs. the low-FZP group (P<0.05). #: There was a significant increase in temporal changes within each group (P<0.05). ♭: There was a significant decrease in temporal changes within each group (P<0.05). WBC: white blood cells, Ht: hematocrit, PLT: platelet, ALB: albumin, ALP: alkaline phosphatase, T-Bil: total bilirubin, CREA: creatinine. The biochemical test results were not measured at T15, T30, and T45. For other complete blood cell count and biochemical test results, please refer to Supplementary File 4.

Figure 1 summarizes the changes in the concentrations of major inflammatory cytokines. Following LPS administration, serum IL-6 levels in the high-FZP group were significantly lower than those in the control group (P<0.01), with significant differences observed after T120. However, plasma concentrations of IL-1, IL-4, IL-8, IL-10, IFN-α, IFN-γ, and TNF-α did not differ significantly across groups.

Fig. 1.

Temporal changes in Interleukin (IL)-1β, IL-6, IL-10, and Tumor Necrosis Factor (TNF)-α following lipopolysaccharide (LPS) administration. The x-axis represents the time elapsed after LPS administration, while the y-axis shows the values of IL-1β, IL-6, IL-10, and TNF-α. In the box plot, the whiskers represent the range of the data, with the upper whisker showing the maximum value and the lower whisker showing the minimum value. The top edge of the box indicates the 75th percentile, while the bottom edge indicates the 25th percentile. The horizontal line within the box represents the median value. In the case of IL-6, the high-dose fuzapladib (FZP) group exhibited significantly lower values than the control group at 120 to 240 min post-LPS administration (*: P<0.05). For other cytokine results, please refer to Supplementary File 5.

During the observation period from T0 to T240, plasma fuzapladib concentrations ranged (min-max) from 0.14 to 0.27 μg/mL in the low-FZP group and from 3.89 to 9.71 μg/mL in the high-FZP group.

DISCUSSION

In this study, administering LPS to pigs successfully replicated a septicemia-like endotoxemia model characterized by a decrease in circulating blood volume and an increase in EVLW, which indicates increased vascular permeability. Although there were no significant effects on complete blood cell count or biochemical tests following fuzapladib administration, high-dose fuzapladib suppressed the elevation of IL-6, a key inflammatory cytokine, and mitigated the transient blood pressure drop and PaO₂ reduction observed after LPS administration. This suggests that the process that led to death during the monitoring period may have been delayed.

Pigs are widely used as experimental animals in cardiovascular research, and we previously investigated endotoxemic porcine models to assess cardiovascular function [5, 6]. Studies using a low dose of LPS (40 µg/kg over 30 min), showed temporary increases in systemic vascular resistance and decreases in cardiac output, but no significant changes in HR or MAP were found [6]. In contrast, studies using a high dose of LPS (160 µg/kg/hr over 75 min) resulted in severe models that consistently developed ARDS, with many pigs dying from hypotension and hypoxemia within 4 hr [5]. Our study aimed to create a model with hypotension and cardiac dysfunction similar to the pathophysiology of clinically encountered sepsis. We set an intermediate dose of LPS (80 µg/kg over 30 min) based on these reports and compared and investigated the effects of fuzapladib on the suppression of acute-phase inflammatory cytokine production and the maintenance of cardiorespiratory function. As a result, we observed transient increases in systemic vascular resistance and decreases in cardiac output in all groups immediately after LPS administration. Subsequently, there were increases in heart rate and decreases in stroke volume and blood pressure, resulting in hypotension and hypoxemia, with some pigs dying, yielding intermediate outcomes. Hemodynamic studies in sheep with continuous low-dose LPS infusion reported immediate secretion of vasoconstrictive substances such as thromboxane A2, resulting in increased peripheral vascular resistance and transient decreases in cardiac output, which progressed to a hypodynamic circulatory state with fatal outcomes [7, 18], which is consistent with our findings. The production of the acute-phase inflammatory cytokine IL-6 was suppressed in the high-dose fuzapladib group, and early circulatory changes after LPS administration were controlled when compared to other groups, suggesting a potential suppression of progression to ARDS and death.

Regarding the cardiovascular system, a significant decrease in MAP was observed over time after LPS administration. Because MAP is determined by CO and SVR, the primary cause of hypotension is thought to be a decrease in CO. In addition, HR and SV modulate CO. In this experiment, HR increased in all groups, whereas SV decreased. Because SV is determined by preload, afterload, cardiac contractility, and cardiac diastolic function, the decrease in SV could be attributed to a decrease in preload indicated by GEDV and contractility indicated by dPmx, as well as a shortened diastolic period due to increased HR. Septic myocardial dysfunction is common in patients with septic shock, indicating a correlation between decreased dPmx levels and worsening sepsis severity [19]. A rise in EVLW, indicating pulmonary edema, and an increase in PVPI both contribute to the decreased preload. The glycocalyx, a glycoprotein that covers the luminal surface of endothelial cells, regulates vascular permeability, leukocyte migration, and albumin repulsion from the vascular wall owing to its negative charge [4, 8]. Sensitized endothelial cells exposed to LPS shed their glycocalyx, exposing large pores through which albumin and fluid leak [3, 34]. However, red blood cells do not leak unless vascular collapse occurs, which causes hemoconcentration within the vessels. Despite an increase in hematocrit in this study, a decrease in ALB levels indicates enhanced vascular permeability caused by LPS. This study confirmed the peripheral circulatory failure associated with endotoxemia, with a rise in PVPI and decreases in CO, GEDV, and MAP. Blood gas analysis revealed decreases in PaO₂, pH, B.E., and HCO₃⁻, as well as an increase in plasma lactate levels across all groups. Tissue hypoxia enhanced glycolysis and lactate accumulation, resulting in an increase in anion gap and decrease in B.E. and HCO₃⁻, indicating lactic acidosis [10]. Although plasma lactate levels are not a direct measure of tissue perfusion, they are commonly used to assess anaerobic metabolism. The International Consensus Definition for Sepsis and Septic Shock (Sepsis-3), recommends using plasma lactate measurement to diagnose septic shock based on cellular function and metabolic abnormalities [24, 28]. The decrease in CO in this study suggests a reduction in oxygen supply to tissues. Cells that have been sensitized to LPS may experience mitochondrial dysfunction, resulting in decreased oxidative phosphorylation and increased glycolysis [17], which leads to lactate buildup and the development of lactic acidosis.

A negative correlation exists between the EVLW and oxygenation capacity in patients with ARDS [26]. The criteria for ARDS in human [1] and veterinary medicine [31] are as follows: PaO₂ <200 mmHg (arterial oxygen partial pressure/fraction of inspired oxygen [P/F] <200) in a 100% oxygen environment. In the current study, elevated EVLW and decreased PaO₂ in many of the test pigs indicated the presence of ARDS. Although no statistically significant differences were observed during this study, PaO₂ <200 mmHg was observed in three pigs in the control group, four pigs in the low-FZP group, and one pig in the high-FZP group. At 45 and 60 min post-LPS administration the high-FZP group had significantly higher PaO₂ levels than the control and low-FZP groups. Fuzapladib has been shown to attenuate lung injury in rats with cerulein-induced pancreatitis complicated by endotoxemia [14]. In humans, normal EVLW levels associated with PaO₂ are approximately 7 mL/kg, but in pulmonary edema the levels exceed 10 mL/kg and severe pulmonary edema exceeds 15 mL/kg [27]. EVLW is associated with mortality due to pneumonia and ARDS [21]. In the ICU, mortality rates are reported to be 60% for patients with an EVLW of 14–21 mL/kg and 65% for those with an EVLW exceeding 21 mL/kg [21]. Although none of the pigs in this study achieved an EVLW of 21 mL/kg, four pigs exceeded 14 mL/kg (one in the control group, two in the low-FZP group, and one in the high-FZP group), and three died during the observation period in the control and low-FZP groups. The high-FZP group had higher initial and sustained blood pressure than the control group, and the absence of a ventilation-perfusion mismatch was deemed a factor in maintaining PaO₂. Thus, in this investigation, the efficacy of high dose fuzapladib to reduce the drop in PaO₂, a sign of lung injury, may have delayed the progression to VetARDS criteria during the observation period. These results suggest that high doses of fuzapladib may prolong the process leading to mortality during endotoxemia by suppressing early post-LPS administration vasodilation. The high-FZP group had a lower incidence of VetARDS. However, the possibility of a type-II error owing to the small number of pigs in the study cannot be discounted.

Endotoxemia is a pathological condition of sepsis, and in human medicine, the Sequential Organ Failure Assessment (SOFA) score is used to assess multiple organ failure [12]. The SOFA score measures the degree of dysfunction in multiple organs, indicates the severity of organ dysfunction, and predicts the prognosis of patients with sepsis. The SOFA score evaluates PaO₂, PLT, T-Bil, MAP, vasopressor dosage, consciousness level, CREA, and urine output [24]. Based on the findings of this study, and excluding vasopressor dosage, consciousness level, and urine output of the SOFA score, evaluation of the SOFA score components revealed that, while not directly applicable between humans and pigs, LPS administration resulted in decreased PaO₂, MAP, and PLT and increased T-Bil and CREA levels in all groups. Elevated levels of ALP, γ-GTP, and TBA, together with increased CREA and BUN, indicated organ hypoperfusion, microthrombi, tissue hypoxia, or renal vasoconstriction due to increased renin-angiotensin system activity, resulting in acute liver and kidney injury [22, 32]. The high-FZP group showed significant suppression of transient increases in CREA, ALP, and γ-GTP, although these were not clinically significant. These findings suggest that high doses of fuzapladib may delay early acute cardiorespiratory dysfunction post-LPS administration, mitigate progression to organ failure, and potentially delay initial LPS responses.

Further investigation revealed significant increases in hematocrit and decreases in WBC and PLT counts over time in all groups. The decrease in WBC and PLT counts is a common observation in sepsis [2], and it has been attributed to the removal of white blood cells from circulation due to their adherence to damaged endothelial cells following LPS administration. Based on the results of complete blood cell count and blood chemistry tests, this study model established a pathological state similar to human sepsis, demonstrating that a sepsis-like condition could be successfully replicated in the endotoxemia porcine model. Fuzapladib was expected to reduce leukocyte adhesion to the injury site; however, in this study, no significant differences in leukocyte trends were identified among the groups, which contradicted predictions. Fuzapladib inhibits LFA-1 activation [23], but in vitro evaluation tests using genetically engineered mouse pre-B cells with LFA-1 introduction and stimulation with stromal cell-derived factor 1 (SDF-1) required a 10 min preincubation, whereas our fuzapladib administration was only for 5 min before LPS administration, potentially insufficiently inhibiting LFA-1 expression due to LPS administration. To assess the inhibitory effect on leukocyte adhesion to endothelial cells, it is considered necessary to administer fuzapladib earlier to confirm its preventive effects.

In terms of inflammatory cytokines, the high-FZP group showed a substantial drop in IL-6 levels from 120 to 240 min compared to the control group. While the low-FZP group did not achieve effective blood concentrations, resulting in no significant difference in IL-6 levels, it was speculated that inhibiting IL-6 production in the high-FZP group could suppress the initiation of inflammatory reactions and thus prevent organ damage. The model used in this study induced inflammation and caused mortality in pigs in less time than in previous reports, and the observation period was set at 4 hr. Although IL-6 expression, an early inflammatory cytokine, was suppressed, there may have been no subsequent protective effects on organ damage or changes in other cytokines across the groups. Steiner et al. undertook a multicenter randomized controlled trial with fuzapladib (0.4 mg/kg IV, SID) in dogs with clinical indications of acute pancreatitis and serum canine pancreatic lipase immunoreactivity (cPLI) values of 400 μg/L or higher. They reported slightly higher clinical activity scores but found no significant differences in cytokine levels, including IL-6. In contrast, our study observed suppression of IL-6 at high doses. This suggests that higher doses of fuzapladib should be administered earlier in diseases requiring systemic inflammation, such as pancreatitis, SIRS, and sepsis.

A limitation of this study is the minimum number of five pigs per group, which was predetermined based on the sample size. The presence of deceased pigs could have resulted in insufficient statistical power and Type I and Type II errors. In the future, it will be necessary to refine the experimental techniques, increase the sample size, and adjust the LPS administration rate to extend the observation period. This re-evaluation is crucial to thoroughly assess the efficacy of fuzapladib in sepsis models.

In conclusion, this study successfully replicated a sepsis model in pigs using Escherichia coli derived LPS. The initial administration of high-dose fuzapladib did not result in hypotension and maintained the ventilation-perfusion ratio, stabilizing PaO₂ levels and preventing the onset of VetARDS in most pigs. While high-dose fuzapladib did not suppress organ damage in the LPS-induced sepsis model, it did reduce the production of IL-6, an inflammatory marker. This suggests that the progression to death could be delayed during the observed period. Fuzapladib is thought to have the ability to slow the progression of systemic inflammatory diseases, such as the endotoxemia model in pigs, if higher doses are administered early on.

CONFLICT OF INTEREST

Ishihara Sangyo Kaisha Ltd., provided the fuzapladib formulation utilized in this study, while Chikako Yoshida and Hiroshi Shikama of the same company measured the fuzapladib blood levels.