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. Author manuscript; available in PMC: 2025 Jun 1.
Published in final edited form as: Arch Toxicol. 2024 Mar 29;98(6):1843–1858. doi: 10.1007/s00204-024-03725-2

The Thrombopoietin Mimetic JNJ-26366821 Reduces the Late Injury and Accelerates the Onset of Liver Recovery After Acetaminophen-induced Liver Injury in Mice

Olamide B Adelusi 1, Jephte Y Akakpo 1, Gary Eichenbaum 2, Ejaz Sadaff 2, Anup Ramachandran 1, Hartmut Jaeschke 1
PMCID: PMC11210275  NIHMSID: NIHMS2004119  PMID: 38551724

Abstract

Acetaminophen (APAP)-induced hepatotoxicity is comprised of an injury and recovery phase. While pharmacological interventions, such as N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP), prevent injury there are no therapeutics that promote recovery. JNJ-26366821 (TPOm) is a novel thrombopoietin mimetic peptide with no sequence homology to endogenous thrombopoietin (TPO). Endogenous thrombopoietin is produced by hepatocytes and the TPO receptor is present on liver sinusoidal endothelial cells in addition to megakaryocytes and platelets, and we hypothesize that TPOm activity at the TPO receptor in the liver provides a beneficial effect following liver injury. Therefore, we evaluated the extent to which TPOm, NAC or 4-MP can provide a protective and regenerative effect in the liver when administered 2h after an APAP overdose of 300 mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12h after APAP. In contrast, TPOm treatment was beneficial at 24h, i.e., all injury parameters were reduced by 42–48%. Importantly, TPOm enhanced proliferation by 100% as indicated by PCNA-positive hepatocytes around the area of necrosis. When TPOm treatment was delayed by 6h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2h after APAP significantly attenuated all injury parameters at 24h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Keywords: Acetaminophen hepatotoxicity, JNJ-26366821, acute liver failure, regeneration, fomepizole, N-acetylcysteine

Introduction

Acetaminophen (APAP) is a common analgesic and antipyretic that is used globally, including by an estimated 60 million Americans every week (Agrawal and Khazaeni 2023). While generally well tolerated at therapeutic doses, overdoses of APAP can cause severe acute liver injury which can progress to liver failure and possibly death (Yoon et al. 2016) and account for over 56,000 annual emergency room visits in the US (Nourjah et al. 2006). APAP overdose is the leading cause of drug-induced liver injury and acute liver failure in the US, accounting for nearly 50% of cases of the latter (Larson et al. 2005; Lee 2017).

Decades of research have resulted in a solid understanding of the pathophysiology of APAP induced liver injury (Jaeschke, et al. 2021b; Ramachandran and Jaeschke 2019). The initiating event is the accumulation of the toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), from cytochrome P450 (particularly CYP2E1 and CYP3A4) catalyzed metabolism of APAP (Jollow et al. 1973; Laine et al. 2009; Lee et al. 1996; Mitchell et al. 1973). At therapeutic doses, NAPQI is eliminated by conjugation to hepatic glutathione (GSH). However, at overdoses, GSH stores are rapidly depleted leaving NAPQI to react with alternate targets, i.e., sulfhydryl groups on proteins (Jollow et al 1973; Mitchell et al. 1974). NAPQI is a highly reactive electrophile which forms adducts with various proteins in the cell including mitochondrial proteins, which impairs their function (Cohen et al. 1997). Mitochondrial protein adducts formation triggers initially superoxide formation by complex III towards the cytosol, which activates a redox-sensitive mitogen-activated protein kinase cascade resulting in the phosphorylation of c-jun N-terminal kinase (JNK) (Nguyen et al. 2021). Phospho-JNK (pJNK) translocates to mitochondria (Hanawa et al. 2008) and amplifies the mitochondrial oxidant stress and peroxynitrite formation (Nguyen et al. 2021; Saito et al. 2010a). The iron-dependent nitration of mitochondrial proteins triggers the mitochondrial permeability transition pore (MPTP) opening (Adelusi et al. 2022; Kon et al. 2010). As a result of the MPTP-induced matrix swelling and outer membrane rupture, intermembrane proteins such as endonuclease G and apoptosis-inducing factor (AIF) are released from the mitochondria into the cytosol, from where they translocate to the nucleus and cause DNA fragmentation and subsequent hepatocyte necrosis (Bajt et al. 2006). The pattern of hepatocyte necrosis in APAP overdose-induced liver injury is distinctly centrilobular due to a combination of lower GSH levels and higher CYP2E1 expression making these hepatocytes uniquely susceptible to injury from APAP overdose (Kennedy et al. 2019; Ma et al. 2020; Umbaugh et al. 2021). Following these events, the release of damage-associated molecular patterns (DAMPs) from necrotic hepatocytes triggers the activation and recruitment of immune cells (Jaeschke et al. 2012; Kubes and Mehal 2012), a critical step in the initiation of liver recovery from the injury (Dambach et al. 2002; Holt et al. 2008; Yang et al. 2019). While the precise triggers and mediators of the recovery phase have not been as thoroughly elucidated as the injury phase, it is known that immune cell infiltration, reestablishment of liver zonation, and hepatocyte proliferation play a critical role in the process (Bhushan et al. 2017; Chauhan et al. 2020; Holt et al. 2010; Hu et al. 2022).

The elucidation of the mechanistic underpinnings of APAP-induced liver injury led to the discovery and application of the only clinically approved antidote, N-acetylcysteine (NAC) (Mitchell et al. 1974; Rumack and Peterson 1978). NAC administration promotes GSH resynthesis (Corcoran and Wong 1986), resulting in improved scavenging of NAPQI (Corcoran et al. 1985) and later peroxynitrite (Saito et al. 2010b). As a result, early (within eight hours of the overdose) administration of NAC can mitigate the occurrence of liver injury after APAP overdose in humans, with its efficacy progressively declining with time in later presenting patients (Licata et al. 2022; Smilkstein et al. 1988). Another prospective therapeutic, fomepizole (4-MP) which is currently in trials as an adjunct to NAC, also acts by arresting the early events in the injury (JNK activation and NAPQI formation) (Akakpo et al. 2022; Kusnik et al. 2022; Link et al. 2022). Unfortunately, agents which target later events in the injury process are presently lacking, and thus, the prospect of a late acting therapeutic which can promote recovery beyond the therapeutic window of NAC remains elusive.

Thrombopoietin mimetics have long been used for treatment of conditions such as immune thrombocytopenia (Hitchcock and Kaushansky 2014). Thrombopoietin is an important hematopoietic cytokine which is the primary regulator of platelet production (Thon and Italiano 2010). It is constitutively produced primarily in the liver and released into the circulation. TPO binds to the thrombopoietin receptor on megakaryocytes and megakaryocyte progenitors, promoting their differentiation and proliferation to platelet maturation, causing an overall increase in platelet number (Kuter 2013). The thrombopoietin receptor is also present on platelets, where ligand binding modulates platelet activation (Chen et al. 1995), as well as on hematopoietic stem cells and myeloid and erythroid progenitors where it mediates a myriad of functions including expansion and maintenance of hematopoietic progenitor cells, mobilization of hematopoietic precursor cells into peripheral blood, and production of vascular endothelial growth factor, VEGF, in hematopoietic stem cells (Chen et al. 1995; Kaushansky et al. 1996; Kirito and Kaushansky 2005; Torii et al. 1998). Disruption in thrombopoietin signaling is known to occur in thrombocytopenia of varying etiologies, inflammatory disorders, and liver failure (Ghanima et al. 2019; Schiødt 2003). In addition to its role in stimulating production of megakaryocytes and platelets, it has also shown effects on liver sinusoidal endothelial cells and hepatocytes following hepatectomy (Hitchcock et al. 2021; Miyakawa et al. 2015). JNJ-26366821 (also referred to as TPOm), is a novel PEGylated thrombopoietin (TPO) mimetic peptide and member of the Thrombopoietin mimetic drug class, which has been found to be safe in animals and humans (Knight et al. 2011; Liem-Moolenaar et al. 2008). In this work we have evaluated the impact of JNJ-26366821 compared to N-acetylcysteine and 4-methylpyrazole (4-MP) on liver injury and recovery following APAP overdose in a mouse model.

Materials and methods

Animals.

All experiments were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Centre and followed the criteria of the National Research Council for the care and use of laboratory animals. Male C57BL/6J mice 8–10-weeks old (Jackson Laboratories, Bar Harbor, Maine) were kept in an environmentally controlled room under a 12-hour light/dark cycle with food and water ad libitum.

Experimental Design.

Acetaminophen, N-acetylcysteine and Fomepizole (4-MP) were obtained from Sigma-Aldrich (St. Louis, Missouri). JNJ-26366821 was generously provided by Johnson and Johnson Consumer Health (New Brunswick, New Jersey). Overnight fasted mice were injected intraperitoneally (i.p.) with 300 mg/kg APAP. After either 2 or 6 hours, the mice were given 1 mg/kg TPOm subcutaneously (s.c.), 500 mg/kg NAC i.p., 50 mg/kg 4-MP i.p. or vehicle (s.c. saline). Food was returned to the mice 6 hours after APAP and the mice were sacrificed 6, 12 or 24 hours after APAP under isoflurane anesthesia. To evaluate the effect of TPOm on APAP metabolism, overnight fasted mice were treated with TPOm 1 mg/kg s.c. or saline 1 hour before APAP 300 mg/kg i.p. Mice were then sacrificed 2 hours after APAP under isoflurane anesthesia. Blood was collected from the inferior vena cava into a heparinized syringe and centrifuged at 20,000 g for 2 minutes to isolate plasma. Livers were collected and rinsed in saline before being sectioned into pieces which were either snap frozen in liquid nitrogen or fixed overnight in 10% phosphate buffered formalin. Hepatic glutathione (GSH) levels were measured in snap frozen tissue using a modified Tietze assay as described in detail (McGill and Jaeschke 2015).

APAP-Protein Adducts Measurement.

Snap frozen liver tissues were homogenized in 10 mM sodium acetate (pH 6.5) using a blade-type homogenizer. Supernatants were collected after centrifugation of the homogenate at 16,000 g for 5 minutes. To remove low molecular weight compounds which might interfere with the measurement, the supernatants were filtered through Bio-Spin 6 columns (Bio-Rad, Hercules, CA), which were pre-washed with 10 mM sodium acetate. The filtrates were subjected to overnight protease digestion to separate the APAP-Cys adducts derived from proteins which were then precipitated using ice-cold 40% trichloroacetic acid (Sigma Aldrich, St. Louis, Missouri). The supernatant was collected and filtered through microcentrifuge tubes. APAP-CYS was then measured using HPLC with electrochemical detection as described (McGill et al. 2012).

Histology and TUNEL Staining.

Tissue was fixed in 10% phosphate buffered formalin and embedded in paraffin before being cut into 5 μm thick sections. Hematoxylin and eosin (H&E) staining was performed on sections to visualize the areas of necrosis. TUNEL staining was performed on rehydrated liver sections using the In Situ Cell Death Detection Kit (Roche, 11684809910) according to the manufacturer’s instructions. Images were then acquired with an Olympus BX51TF microscope and necrotic areas were quantified on three randomly selected 100X images using ImageJ (Schindelin et al. 2012).

Immunohistochemistry and Immunofluorescence.

Rehydrated tissue sections underwent heat mediated antigen retrieval using a citrate buffer (pH 6.0). The sections were blocked in 3% BSA in serum before being incubated overnight with anti-rabbit PCNA (Cell Signaling Technologies, #13110, 1:12000). Endogenous peroxidases were quenched by incubation in 3% hydrogen peroxide for 20 minutes. The tissue sections were then incubated in SignalStain Boost IHC Detection Reagent (HRP, Rabbit) (Cell Signaling Technologies, #8114) for 30 minutes. SignalStain DAB Substrate Kit, (Cell Signaling Technologies #8059) or Alkaline Phosphatase Substrate Kit (Vector, SK-5100) were used as detection reagents. The slides were imaged using an Olympus BX51TF microscope and the numbers of PCNA-positive cells were counted using QuPath-0.3.0 (Bankhead et al., 2017). For immunofluorescence, heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0) and sections were incubated overnight with anti-rabbit Cytochrome P450 2E1 (Abcam, ab28146, 1:200) and anti-mouse Cytochrome P450 2F2 (Santa Cruz Biotechnology, sc-374540, 1:400). The sections were incubated in the following secondary antibodies for 30 minutes: Alexa Fluor 488 Goat anti-mouse IgG (BioLegend, #405319, 1:500) and Alexa Fluor 594 Goat anti-Rabbit IgG (ThermoFisher, A-11012 1:500). Imaging was done using a Nikon Eclipse Ti2 microscope. CYP2E1- and CYP2F2-positive areas were quantified using ImageJ and the ratio of CYP2E1 positive to CYP2F2 positive areas was determined.

Western Blotting.

Western blotting was performed on snap frozen tissue as described (Adelusi et al. 2022). The following primary and secondary antibodies were obtained from Cell Signaling Technologies (Danvers, MA) as follows: anti-rabbit JNK (1:1000, #9252), anti-rabbit p-JNK (1:1000, #9251), anti-rabbit PCNA (1:1000, #13110) and anti-rabbit GAPDH (1:1000, #5174), anti-rabbit IgG, HRP-linked antibody (1:5000, #7074). Proteins were visualized using chemiluminescence detecting reagents (Cytiva, Marlborough, Massachusetts) on a LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, Nebraska). Densitometric analysis was performed on LI-COR Image Studio Lite (LI-COR Biosciences) by quantifying the intensities of individual bands and normalizing to the intensity of the loading controls.

Statistical Analysis.

Comparisons between two groups were performed using the Student’s t-test. Comparisons between more than two groups were made using one way analysis of variance (ANOVA) followed by a post-hoc Dunnett’s test. P values < 0.05 were taken to be statistically significant. Statistical analysis was performed on GraphPad Prism version 8.0.1 for Windows (Graph-Pad Software, San Diego, California).

Results

Characterization of the effect of JNJ-26366821 on APAP metabolism and initiation of injury after APAP overdose

When investigating any compound as a possible therapeutic against APAP overdose, it is vital to investigate its effect on the initiating events in the pathophysiology to determine its viability as a therapeutic for clinical use (Jaeschke et al. 2021a). Cytochrome P450-mediated metabolic activation of APAP to NAPQI is the first step in the pathophysiology, without which injury would not occur. Therefore, we determined if JNJ-26366821 affects this process by measuring APAP protein adducts in the liver, a reliable indicator of NAPQI formation and by extension oxidative APAP metabolism. To do this, fasted mice were given a pretreatment with JNJ-26366821 1 hour before a 300 mg/kg dose of APAP and protein adducts in the liver were measured 2 hours after APAP, which represents the peak of adducts formation (McGill et al., 2013). JNJ-26366821 treated mice had similar levels of hepatic protein adducts (APAP-Cys) at 2 hours (Figure 1A), indicating that it does not affect APAP oxidative metabolism. This inference is supported by the fact that the recovery of hepatic total glutathione (GSH) and the increase in glutathione disulfide (GSSG) levels remain unchanged compared to vehicle at 6 hours when JNJ-26366821 was administered 2 hours after APAP (Figure 1B–D). This suggests no impact on GSH replenishment or oxidative stress generation by JNJ-26366821 at 6 hours after APAP overdose. To further verify that JNJ-26366821 does not affect oxidative stress after APAP overdose, we looked at JNK phosphorylation at 6 hours as JNK activation (triggered by cytosolic oxidant stress) and subsequent mitochondrial translocation of P-JNK are critical in setting off the mitochondrial dysfunction which ultimately results in hepatocyte necrosis. Unsurprisingly, based on the adduct and GSH data, there was no difference in JNK activation between mice treated with vehicle or JNJ-26366821 (Figure 1E).

Figure 1: JNJ-26366821 (TPOm) does not inhibit APAP metabolism or oxidative stress.

Figure 1:

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Overnight fasted mice were treated with 1 mg/kg TPOm or vehicle 1 hour before 300 mg/kg APAP. (A) APAP-Cys adduct levels at 2 hours after APAP overdose. In a second experiment, overnight fasted mice were treated with 1 mg/kg TPOm or vehicle 2 hours after 300 mg/kg APAP. (B) Glutathione disulfide (GSSG) concentration, (C) Total glutathione concentration (D) Ratio of GSSG to total GSH and (E) Immunoblot showing JNK activation at 6 hours after APAP overdose. Bars represent mean ± SEM for n=3–6 mice.

After confirming that JNJ-26366821 does not affect NAPQI formation, GSH resynthesis or oxidative stress generation, we looked directly at markers of liver injury at 6 and 12 h after APAP, which mark the peak of the early injury phase and the beginning of the late injury phase respectively (Jaeschke et al., 2021a). We observed the progression of liver injury from 6 to 12 h after APAP with increase in all injury markers (plasma ALT activity, area of necrosis and DNA fragmentation as delineated by TUNEL staining) (Figure 2A–E). However, there were no significant differences in these parameters with JNJ-26366821 intervention, indicating that JNJ-26366821 confers no protection from early injury initiation after APAP overdose.

Figure 2: TPOm does not prevent induction of liver injury up to 12 hours after APAP overdose.

Figure 2:

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(A) Plasma alanine amino transferase (ALT) activity, (B) necrotic area quantification, (C) quantification of TUNEL stained area and, (D) representative H & E and (E) TUNEL staining images at 6 and 12 hours after APAP overdose. Bars represent mean ± SEM for n=4–6 mice.

JNJ-26366821 attenuates late injury and accelerates the onset of liver recovery 24 hours after APAP overdose

In this mouse model of APAP overdose-induced liver injury, 24 h after APAP demarcates the end of the injury phase and the start of the recovery phase for a dose of 300 mg/kg (Bhushan et al. 2014). When we studied mice treated with JNJ-26366821 2 h after APAP at this time point, we observed a striking contrast to the outcomes obtained at the 6 and 12 h time points. Injury parameters were markedly blunted in mice that received JNJ-26366821. Mean plasma ALT levels were lower in the JNJ-26366821 treated group compared to vehicle, although the difference did not reach statistical significance (Figure 3A). Furthermore, average area of necrosis was significantly reduced from 40% in vehicle treated mice to 18% with JNJ-26366821 treatment (Figure 3B,D). In addition, the TUNEL positive area was also significantly reduced in JNJ-26366821 treated mice (Figure 3C,E).

Figure 3: TPOm treatment reduced liver injury at 24 hours after APAP overdose.

Figure 3:

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Overnight fasted mice were treated with 1 mg/kg TPOm or vehicle 2 hours after 300 mg/kg APAP. (A) Serum alanine amino transferase (ALT) activity, (B) necrotic area quantification (C) quantification of TUNEL stained area and, (D) representative H & E and (E) TUNEL staining images 24 hours after APAP. Bars represent mean ± SEM for n=4–6 mice. *p<0.05 compared to vehicle.

In healthy livers, the liver lobule exhibits metabolic zonation with pericentral and periportal hepatocytes being highly functionally specialized and genetically distinct (Jungermann, 1986; Umbaugh et al. 2021). Pericentral hepatocytes are characterized by expression of CYP2E1 and are selectively vulnerable to injury from APAP overdose while periportal hepatocytes, on the other hand, are characterized by expression of CYP2F2 (Roberts et al. 1991; Umbaugh et al. 2021). The progressive loss of zonation and pericentral, CYP2E1-positive hepatocytes is characteristic of APAP-induced liver injury. To track how JNJ-26366821 affects this process, staining was performed on liver sections to visualize periportal (CYP2F2 positive) and pericentral (CYP2E1 positive) hepatocytes, respectively, from 6 to 24 h after APAP and the areas of CYP2E1 and CYP2F2 staining were quantified. Untreated control livers had nearly equal amounts of periportal and pericentral hepatocytes with CYP2E1-positive areas being nearly 50% (Figure 4). While there was no decrease in CYP2F2 periportal hepatocytes from control levels after APAP, there was a decline in CYP2E1 positive areas to 36.6% and 31.8% at 12 h after APAP in vehicle and JNJ-26366821 treated groups, respectively, indicating a progressive loss of CYP2E1 positive (pericentral) hepatocytes (Figure 4). There was no statistically significant difference in CYP2E1 areas between vehicle and JNJ-26366821 treated mice up to 12 h post APAP. Remarkably, however, we observed clear differences at 24 h after APAP. While APAP treated mice only showed a few surviving CYP2E1-positive hepatocytes at 24h (Figure 4-yellow arrows), JNJ-26366821 treatment caused statistically greater CYP2E1 expression in comparison to vehicle at 24 h after APAP. While CYP2E1-positive pericentral hepatocytes declined dramatically in the vehicle treated group from 36.6% at 12 h to 14.1% at 24 h, CYP2E1 expression was nearly steady in the JNJ-26366821 treated group with a nominal decrease from 31.8% at 12 h to 29.4% at 24 h (Figure 4). Thus, there were significantly more pericentral hepatocytes in JNJ-26366821 treated mice at 24 h than in vehicle treated mice. This indicates that JNJ-26366821 treatment prevents the loss of CYP2E1-positive pericentral hepatocytes beyond 12 h after APAP and promotes the maintenance of liver zonation.

Figure 4: Preservation of liver zonation by TPOm at 24 hours after APAP overdose.

Figure 4:

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Representative images of CYP2E1 (red) and CYP2F2 (green) immunofluorescent staining and line graph showing the percent of the CYP2E1 positive area compared to the total area (CYP2E1+CYP2F2). Points represent mean ± SEM for n=4 mice. *p<0.05 compared to APAP + vehicle.

As the 24 h time point marks the beginning of the recovery phase in the liver (Bhushan et al. 2014), we determined if JNJ-26366821 can alter this process because hepatocyte proliferation to replace necrotic hepatocytes is essential for liver repair after APAP overdose (Clemens et al. 2019). In addition, expression of PCNA as marker for proliferating hepatocytes, was shown to be higher in acute liver failure patients who survived compared to non-survivors (Kayano et al. 1992). Thus, to determine the effect of JNJ-26366821 on liver recovery after APAP overdose, we evaluated the expression of PCNA 24 hours after APAP. Immunostaining for PCNA 24 h after APAP revealed a significantly greater number of PCNA positive cells after JNJ-26366821 treatment around the area of necrosis than after vehicle treatment (Figure 5A). Western blotting for PCNA recapitulated these findings, with a trend towards higher PCNA expression in JNJ-26366821-treated mice versus vehicle (Figure 5B). However, the western blot data represent the average of the entire liver while the immunostaining clearly shows that cells around the area of necrosis are the main proliferating cells (Figure 5A), which eventually replace the dead cells in the centrilobular area (Nguyen et al. 2022). These results indicate a more robust recovery response in JNJ-26366821-treated mice when compared to vehicle.

Figure 5: Enhanced hepatocyte proliferation with TPOm treatment at 24 hours after APAP overdose.

Figure 5:

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(A) Representative immunohistochemistry images and of proliferative cell nuclear antigen (PCNA) and quantification of PCNA positive cells and, (B) Western blot images and quantitation of PCNA and GAPDH at 24 hours after APAP. Bars represent mean ± SEM for n=4 mice. *p<0.05 compared to vehicle. FOV, field of view.

Comparison of JNJ-26366821 with N-acetylcysteine and fomepizole

Given that JNJ-26366821 has been found to promote recovery at 24 h after APAP, we examined how it compares to well characterized interventions which are currently in clinical use, such as N-acetylcysteine (NAC) and fomepizole (4-MP). To this end, mice were administered 300 mg/kg APAP followed by NAC, 4-MP, JNJ-26366821 or vehicle 2 h after APAP. At 24 h post APAP, all three interventions reduced plasma ALT, areas of necrosis and DNA fragmentation to varying extents with NAC causing the most reduction and JNJ-26366821 and 4-MP having similar magnitudes of effect (Figure 6A–D). However, when we looked at liver recovery at the same time point, JNJ-26366821 distinguished itself from the other therapeutics. Immunostaining for PCNA showed that JNJ-26366821-treated mice averaged a significantly higher number of PCNA positive cells around the areas of necrosis than vehicle treated mice, while NAC and 4-MP did not have this effect (Figure 7A,B).

Figure 6: TPOm, N-acetylcysteine (NAC) and Fomepizole (4-MP) are protective at 24 hours after APAP.

Figure 6:

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Overnight fasted mice were treated with 1 mg/kg TPOm, 500 mg/kg NAC, 50 mg/kg 4-MP or vehicle 2 hours after 300 mg/kg APAP. (A) Plasma ALT, (B) necrotic area quantification, (C) quantification of TUNEL stained area and, (D) representative H & E and TUNEL staining images at 24 hours after APAP overdose. Bars represent mean ± SEM for n=4–6 mice. *p<0.05 compared to vehicle.

Figure 7: Significant increase in hepatocyte proliferation absent with NAC and 4-MP treatment, unlike TPOm.

Figure 7:

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(A) Representative immunohistochemistry images for PCNA and (B) quantification of PCNA positive cells at 24 hours after APAP. Bars represent mean ± SEM for n=3–4 mice. *p<0.05 compared to vehicle

Finally, we determined if delayed treatment with JNJ-26366821 can still have beneficial effects after APAP overdose, particularly in comparison with NAC and 4-MP. Again, fasted mice were treated with 300 mg/kg APAP. In this experiment, however, JNJ-26366821, NAC and 4-MP were administered 6 h after the overdose. When markers of injury were measured 24 h after APAP, none of the interventions were able to reduce these parameters compared to vehicle (data not shown) which is expected since delayed treatment was provided long after injury initiation. However, when recovery was investigated, JNJ-26366821 again showed a distinct advantage. JNJ-26366821 treatment significantly increased the number of PCNA positive cells with JNJ-26366821 treatment compared to vehicle (Figure 8A,B). Neither NAC nor 4-MP had any significant effect on PCNA expression (Figure 8A,B). Therefore, JNJ-26366821 can maintain a pro-proliferative, pro-recovery effect when giving at a delayed time point, despite not having any effect on injury.

Figure 8: Delayed treatment with TPOm retains pro-proliferative effect on hepatocytes.

Figure 8:

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Overnight fasted mice were treated with 1 mg/kg TPOm, 500 mg/kg NAC, 50 mg/kg 4-MP or vehicle 6 hours after 300 mg/kg APAP. (A) Representative immunohistochemistry images for PCNA, and (B) quantification of PCNA positive cells at 24 hours after APAP. Bars represent mean ± SEM for n=3–4 mice. *p<0.05 compared to vehicle.

Discussion

The objective of this study was to characterize the effects of JNJ-26366821 in a clinically relevant murine model of APAP-induced liver injury. Given the mechanistic similarities in APAP hepatotoxicity between humans and mice (Jaeschke et al. 2014), any protection in the mouse model generally translates well to a clinical effect as demonstrated by the antidotes NAC and fomepizole (Akakpo et al. 2022). Our investigations showed that JNJ-26366821 did not affect the early injury phase but mitigated loss of hepatic metabolic zonation and enhanced hepatocyte proliferation by 24 h. Hepatocyte proliferation to replace necrotic hepatocytes is essential for liver repair after APAP overdose (Clemens et al. 2019). Inhibition of hepatocyte proliferation has been demonstrated to retard liver regeneration and recovery, while enhancement of hepatocyte proliferation has been shown to promote liver regeneration (Bhushan et al. 2017; Chanda et al. 1995). Furthermore, expression of PCNA, which marks proliferating hepatocytes, was found to be higher in surviving acute liver failure patients versus non-survivors (Kayano et al. 1992). However, the key issue is that JNJ-26366821 does not promote proliferation of all hepatocytes but affects only the hepatocytes surrounding the area of necrosis, which are known to replace the necrotic cells (Nguyen et al., 2022). Intriguingly, JNJ-26366821 exhibits a pattern of protection which is distinct from interventions targeting early injury such as NAC and 4MP which were not found to enhance proliferation in a similar manner. Furthermore, while NAC and 4MP treatment granted no protection after delayed treatment, JNJ-26366821 still proved advantageous, enhancing hepatocyte proliferation. Its established safety profile and unique beneficial effects make JNJ-26366821 a viable therapeutic candidate for APAP-induced liver injury, possibly as an adjunct to NAC treatment. In particular, stimulation of hepatocyte proliferation even with delayed treatment suggests that JNJ-26366821 may be useful in management of late presenting patients.

Potential mechanisms by which thrombopoietin and JNJ-26366821 act in APAP overdose induced liver injury

Serum thrombopoietin levels have been shown to increase in APAP-induced acute liver failure patients (approximately 2–3 days after presentation to the hospital) (Schiødt et al. 2003). In addition, there is a a decrease in platelet counts in APAP overdose patients with ALF and a greater decrease in platelets is associated with greater liver injury (Fischereder and Jaffe 1994; Larson et al. 2005, Schiødt et al. 2003). However, the exact cause of these observations and how TPO and its receptor impact liver injury and recovery after APAP overdose remains to be investigated. The hemostatic system is known to be involved after APAP-induced liver injury (Ganey et al. 2007; Groeneveld et al. 2023). Furthermore, platelet deposition and activation have been shown to modulate liver injury and regeneration after acetaminophen overdose (Chauhan et al. 2020; Miyakawa et al. 2015). Thrombopoietin and JNJ-26366821 have been shown to protect endothelial cells from death following acute insults (Ashcraft et al. 2018; Mouthon et al. 2001) and the TPO receptor is present on liver sinusoidal endothelial cells (LSECs). Acetaminophen is known to be directly toxic to LSECs in mouse models (DeLeve et al. 1997; McCuskey 2006) and patients (Wang et al. 2020; Williams et al. 2003). Furthermore, LSEC-mediated signaling is essential for the reestablishment of liver zonation and liver regeneration and recovery after acute injury, underlining the vital role played by LSECs in the pathology (Ding et al. 2010; Hu et al. 2022; Ma et al. 2020). Furthermore, thrombopoietin mediated interaction between platelets and endothelial cells have been demonstrated to drive liver regeneration after acute injury (Shido et al. 2017). Taken together, these results suggest that TPO and JNJ-263666821 may cause their beneficial effects on liver regeneration and recovery after APAP-induced liver injury by binding to the TPO receptor on LSECs. Our data showing enhanced liver recovery and maintenance of liver zonation with administration of a thrombopoietin mimetic after APAP overdose provides the first clear evidence that supports this hypothesis. However, more studies are needed to investigate the specific molecular mechanisms of this effect.

JNJ-26366821as a potential therapeutic against APAP overdose induced liver injury

Decades after the introduction of NAC as an antidote, APAP overdose-induced liver injury remains the leading cause of acute liver failure in western countries and the second most common cause of ALF globally (Agrawal and Khazaeni 2023; Rumack and Peterson 1978). This can be attributed, at least in part, to the fact that NAC is most effective when given within 10 hours of the overdose, which is not always achievable in practice. For example, patients who unintentionally ingest an overdose, which account for over half of APAP overdose cases, are more likely to seek medical aid only at the onset of symptoms. Such patients are therefore at risk of poor response to NAC treatment (Larson et al. 2005; Reuben et al. 2016). The time point at which JNJ-26366821 was administered in these experiments (2 hours after APAP) marks the end of APAP metabolism and the beginning of ALT release in mice, corresponding to roughly 24 hours after APAP overdose in humans (McGill et al., 2011; Xie et al., 2014). This makes it potentially beneficial even after the therapeutic window of NAC has elapsed (Akakpo et al., 2021; Smilkstein et al., 1988). While 4-MP, the new antidote under clinical development, appears to expand the therapeutic window of NAC (Akakpo et al. 2022), it targets mechanisms relatively early in the pathophysiology to prevent cell death. In contrast to NAC which supports the scavenging of NAPQI and peroxynitrite by GSH (Saito et al. 2010b), 4MP prevents NAPQI formation by inhibiting Cyp2E1 and prevents peroxynitrite formation by inhibiting JNK (Akakpo et al. 2022). Though these direct effects make 4MP more effective than NAC, treatment with 4MP is fundamentally not a different therapeutic approach. In contrast, JNJ-26366821 did not have the same effects as NAC or 4MP on the injury but impacted the late injury and promoted regeneration.

Summary and Conclusion

Delayed treatment with the thrombopoietin mimetic JNJ-26366821 did not impact early events in the APAP-induced injury process including protein adducts formation, JNK activation and cell necrosis up to 12 h after APAP administration. In contrast, it attenuated the late injury phase, and it promoted regeneration, an effect which was independent of the impact on the late injury. Thus, the effect of JNJ-26366821 in this model is fundamentally different compared to the standard of care NAC and the new antidote in clinical development 4MP (fomepizole), which mainly prevent cell death but does not have a direct impact on regeneration. Therefore, JNJ-26366821 is a potential novel therapeutic for enhancement of liver recovery after APAP-induced liver injury and prevention of acute liver failure.

Acknowledgements.

This work was funded in part by a grant from Johnson & Johnson, Consumer Health and by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01 DK102142 and R01 DK125465, and National Institute of General Medicine (NIGMS) funded Liver Disease COBRE grants P20 GM103549 and P30 GM118247. J.Y.A. was funded by a postdoctoral fellowship from the CTSA grant from NCATS awarded at the University of Kansas for Frontiers: University of Kansas Clinical and Translational Science Institute No. TL1TR002368.

Conflict of interest.

GE and ES were employees of Johnson & Johnson; HJ was the recipient of research grants from Johnson & Johnson Consumer Health; all others have no conflict of interest to declare.

Abbreviations:

AIF

apoptosis-inducing factor

APAP

acetaminophen

GSH

glutathione

H&E

hematoxylin and eosin

JNK

c-jun N-terminal kinase

MPTP

mitochondrial permeability transition pore

NAC

N-acetylcysteine

NAPQI

N-acetyl-p-benzoquinone imine

TPOm

PEGylated thrombopoietin mimetic

TUNEL assay

terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay

Data availability.

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. All data generated or analyzed during this study are included in this published article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. All data generated or analyzed during this study are included in this published article.

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