Treprostinil Improves Hepatic Cytochrome P450 Activity during Rat Liver Transplantation

Abstract

Background

Cytochrome P450 (CYP450) activity is an important indicator of liver graft function. CYP450 activity is altered by pro-inflammatory cytokines, which are associated with ischemia-reperfusion (I/R) injury during orthotopic liver transplantation (OLT). Treprostinil, an FDA-approved prostacyclin analog, ameliorated cold I/R injury during rat OLT. We hypothesized that treprostinil would improve CYP450 activity in liver graft during cold I/R injury post-OLT.

Methods

OLT was performed in syngeneic male Lewis rats with 18 h graft preservation in cold UW solution. Donor and recipients received treprostinil (100 ng/kg/min) or matching placebo for 24 h before and up to 48 h post-OLT. Liver graft mRNA and protein expression of CYP450 isoforms were analyzed by qRT-PCR and Western blot analysis, respectively. The formation rates of 1-hydroxymidazolam and 6β-hydroxytestosterone, 6-hydroxychlorzoxazone, 2α- and 16α-hydroxytestosterone in liver graft microsomes served as markers for CYP3A, CYP2E1, and CYP2C11 activity, respectively, and were measured by LC–MS.

Results

Treprostinil significantly decreased serum ALT and AST levels at 6–48 h after OLT, compared to placebo. The expressions of TNFα and IFNγ mRNA in the liver graft were significantly inhibited in the treprostinil-treated group at 1 h post-reperfusion. Treprostinil restored CYP2E1 protein expression to that of normal liver and significantly improved CYP3A activity to more than two-fold of placebo early post-OLT.

Conclusions

Treprostinil significantly ameliorated hepatic injury, reduced expression of pro-inflammatory cytokines, and improved CYP450 activity in liver graft early post-OLT. These findings suggest that treprostinil has the potential to serve as a therapeutic option to protect liver graft function against I/R injury during clinical OLT.

Orthotopic liver transplantation (OLT) is the only curative therapy available for patients with end-stage liver disease. The surgical procedure of liver transplantation inherently involves cold ischemia during graft preservation ex vivo, followed by reperfusion of the transplanted graft in vivo, which results in varying degrees of cold ischemia and reperfusion (I/R) injury. Cold I/R injury is a major cause of both initial poor function and primary graft non-function, leading to organ dysfunction and early graft failure, which carries a high mortality rate if patients are not re-transplanted immediately. I/R injury greatly contributes to the impaired function of the transplanted liver graft and further depletes the already scarce donor pool. The need to ameliorate I/R injury in liver transplantation is imminent, however, no therapeutic agents are available to prevent I/R injury during OLT.

The activities of cytochrome P450 (CYP450) enzymes are an important indicator of liver graft function in vivo.^1–3 Considering that the liver is the most important site of drug metabolism and clearance, changes in CYP450 activity can directly alter the hepatic clearance of drugs. Consequences of reduced drug clearance include drug toxicity and sub-therapeutic plasma drug concentrations, which could precipitate hepatic dysfunction or lead to graft failure. Cold I/R injury is associated with a pro-inflammatory response that can alter CYP450 activity in the transplanted liver graft. Although various models of inflammation have been used to mimic effects of I/R injury on CYP450 activity^4,5, limited data exist on direct effects of cold hepatic I/R injury on CYP450 expression and activity in a clinically relevant animal OLT model. Different models of inflammation and infection suppress different CYP450 isoforms in vivo.^6,7 Therefore, it is important to quantify the changes in CYP450 regulation in the liver graft post-transplant in a clinically relevant animal OLT model, which incorporates liver graft storage in cold preservation solution, followed by warm reperfusion after vascular reconnection.

Of the various pharmacological agents that have been explored to minimize I/R injury during OLT, the prostaglandin (PG) class of drugs has been evaluated to the greatest extent. PGs have well characterized vasodilatory and anti-platelet aggregatory properties and many analogs, including prostacyclin (PGI₂), have been evaluated for their ability to reduce hepatic I/R injury after OLT. However, poor stability, intolerable side effects, and no apparent difference in primary endpoint have limited their clinical application thus far. Treprostinil (Remodulin®), the most recent FDA-approved PGI₂ analog for treatment of pulmonary arterial hypertension, has a higher stability, potency, and longer elimination half-life than other PGI₂ analogs available⁸, thereby, allowing greater therapeutic concentrations to be achieved with less adverse effects. Recently, we demonstrated that treprostinil ameliorated hepatic I/R injury during rat OLT.⁹ Administration of treprostinil to donor and recipient animals prior to hepatectomy and transplantation significantly reduced neutrophil infiltration and hepatic necrosis, restored ATP levels in liver grafts to normal, preserved the sinusoidal endothelial cell lining and reduced platelet deposition early post-transplantation. Finally, hepatic tissue blood flow, which was significantly compromised in the placebo-treated group, was maintained to near normal values by treprostinil.⁹ In the current study, we examine effects of I/R injury on CYP450 activity and the impact of treatment with treprostinil to prevent I/R injury in a rat OLT model. We hypothesized that treprostinil would reduce hepatic inflammation, thereby resulting in improved CYP450 activity in liver graft tissue post-transplantation. We evaluated this hypothesis using a clinically relevant rat orthotopic liver transplantation model.

Methods

Animals and Orthotopic Liver Transplantation

All experimental procedures were performed according to the guidelines of the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

The use of a syngeneic rat OLT model allows investigation of ischemia and reperfusion injury during liver transplantation and eliminates the risk of immunologic response. Male Lewis rats weighing 200–300 g (Harlan Sprague Dawley, Inc, Indianapolis, IN) were maintained in a laminar flow, specific pathogen–free atmosphere at the University of Pittsburgh with a standard diet and water supplied ad libitum. Basic techniques of liver harvesting and OLT without hepatic arterial reconstruction were performed, as described.¹⁰ Briefly, rats were anesthetized with isoflurane inhalation, a midline incision in the abdominal cavity was made and the donor liver graft was excised and immediately flushed with cold UW solution, stored in a bath of UW solution at 4 °C for 18 h, then orthotopically transplanted into syngeneic recipients. After surgery, animals were kept under a heating lamp for 2 h and were given regular food and water ad libitum. The general condition of the rats was checked three times daily. All surgeries were performed by the same surgeon.

Treprostinil Administration and Experimental Design

Fifty-eight animals were divided into three groups: (1) normal liver (NL), which represents control animals not subjected to surgery, or donor + recipient treatment with (2) treprostinil (100 ng/kg/min) or (3) matching placebo (chemicals are described in Supplementary Material 1). Study drug was administered subcutaneously via an Alzet® osmotic pump (Durect Corp., Cupertino, CA). Donor animals received treprostinil or placebo for 24 h prior to hepatectomy and corresponding recipient animals received study drug for 24 h before engraftment and throughout the study period. Pumps were primed for 18 h prior to implantation and implanted 24 h before donor hepatectomy and recipient engraftment to ensure steady-state concentrations throughout the study period. The dose was selected to achieve target therapeutic concentrations.¹¹ Recipients were sacrificed at 1, 3, 6, and 48 h post-transplantation to study early and late effects of treprostinil on cold hepatic I/R injury. Investigators were blinded to treatment allocation during the study period and individual result analyses.

Parameters of Liver Injury

Blood was collected at 0, 1, 3, 6, 24, and 48 h post-transplantation. Serum bilirubin and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by standard enzymatic methods. Area under the serum ALT and AST concentration time curves include measurements at 0, 6, 24, and 48 h post-OLT (AUC_0–48) and were calculated by non-compartmental analysis, using standard linear trapezoidal method with WinNonlin® software (Pharsight, Mountain View, CA).

Messenger RNA Expression

RNA was extracted from liver graft tissue (50–100 mg) using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Two micrograms of total RNA was used to generate cDNA by use of the First Strand cDNA synthesis kit (Promega, Madison, WI). Hepatic mRNA expression was measured by qRT-PCR performed on an ABI PRISM 7000 Sequence Detection System with TaqMan® primer probe sets for CYP3A1, 3A2, 3A18, 2E1, 2C11, TNF-α, IFN-γ, IL-1β, -6, -10, and GAPDH (Applied Biosystems, Forster City, CA). Samples were analyzed in triplicate and relative gene expression was measured using the comparative C_T method with GAPDH as internal control.

Preparation of Liver Graft Microsomes

Snap-frozen slices of rat liver graft were used to prepare microsomes by a standard differential centrifugation procedure with minor modifications.¹² Briefly, liver pieces were electrically homogenized (Polytron, Brinkmann Instruments, Westbury, NY) with 3 volumes of a homogenization buffer (50 mM Tris–HCl buffer, 1.0% KCl, and 1 mM EDTA, pH 7.4). The homogenate was centrifuged (Optima XL-100K ultracentrifuge, Beckman Instruments, Palo Alto, CA) at 10,000g for 20 min 4 °C and supernatant was further centrifuged at 105,000g for 65 min (4 °C). Microsomes were reconstituted using a manual glass homogenizer (Wheaton, Millville, NJ) in twice their weight with 50 mM Tris–HCl buffer (pH 7.4) containing 20% glycerol. Aliquots were stored at −80 °C. The protein concentration was determined by the Bradford method.¹³

Analysis of Protein Expression

Protein levels of CYP3A2, 2E1, and 2C11 in rat liver graft microsomes were measured by Western immunoblotting. Microsomal protein (25 μg) was separated by SDS-PAGE (10% NuPAGE, Invitrogen, Carlsbad, CA), transferred to a PVDF membrane and blocked overnight in 5% Non-Fat Dry Milk, prepared in 1× TBS containing 0.1% Tween 20. Membranes were probed with rabbit anti-rat CYP3A2, CYP2E1, or CYPC11 antibodies (Abcam, Cambridge, MA) and with HRP-conjugated goat anti-rabbit IgG (Abcam). Immunodetection was performed using an ECL detection kit (Thermo Scientific, Rockford, IL). Protein band density was quantified using Image J software 1.40 (National Institutes of Health, Bethesda, MD) and values were normalized to GAPDH (Abcam).

Analysis of Microsomal Cytochrome P450 Activity

Hepatic CYP450 activity was determined using microsomes prepared from rat liver graft. Time of incubation, protein concentration, and substrate concentration were optimized such that each reaction took place in the linear working range. Each incubation included microsomal protein, 10 mM MgCl_2, and 0.1 mM phosphate buffer, pH = 7.4. Samples were pre-incubated in a shaking water bath at 37 °C for 5 min before addition of 1 mM NADPH. Reactions were terminated by addition of ice-cold methanol and samples were placed on ice. Samples were centrifuged at 3000 rpm for 10 min and analyzed immediately.

Midazolam Assay

MDZ is predominantly metabolized to 1-hydroxymidazolam (1-OH MDZ) by CYP3A1 and CYP3A2 in rats and can be used as a biomarker of CYP3A activity in vivo.¹⁴ Incubations contained 0.375 mg/ml microsomal protein and 300 nM MDZ. Samples were incubated for 20 min in a shaking water bath at 37 °C. The concentration of 1-OH MDZ was determined using an Alliance HPLC (Waters 2695, Milford, MA) attached to a Quatromicro™ mass spectrometer (Waters), operated in positive electrospray ionization.

Chlorzoxazone Assay

The formation rate of 6-hydroxychlorzoxazone (6-OH CZN) from CZN was used as a substrate to measure CYP2E1 activity. Incubations contained 0.75 mg/ml microsomal protein and 200 μM CZN. Samples were incubated for 30 min in a shaking water bath at 37 °C. The concentration of 6-OH CZN was measured using an Alliance HPLC system (Waters 2695, Milford, MA) with a Photodiode Array detector (Waters 2998) set at 297 nm.

Testosterone Assay

The main male-specific rat isoform of CYP450 is 2C11 which gives a high yield of oxidized testosterone TST in 2α- and 16α-positions.^14,15 The formation rate of 2α- and 16α-OH TST was used to measure CYP2C11 activity and the formation rate of 6β-OH TST was used as second substrate to measure CYP3A activity. Incubations consisted of 0.5 mg/ml microsomal protein and 150 μM TST. Samples were incubated for 20 min in a shaking water bath at 37 °C. The concentration of 2α-, 6β-, and 16α-OH TST were individually measured by HPLC-UV. Procedure details described in Supplementary Material 2.

Statistical Analysis

Results are reported as the mean ± SEM from 3 to 6 individual experiments. Student's t-test or one-way analysis of variance was performed to determine the difference between the groups, followed by Bonferroni post-hoc analyses using Prism software (GraphPad, San Diego, CA). Differences were considered significant at a p-value < 0.05.

Results

Clinical Course and Assessment of Tolerability

Following vascular anastomoses, no excessive bleeding in the treprostinil-treated group, relative to placebo, occurred. Bile formation was immediate upon reperfusion of the liver graft in the treprostinil-treated group. Treprostinil-treated animals functioned normally and appeared to recover sooner after surgery than the placebo-treated animals, which appeared weaker throughout the post-OLT study period. No difference in body weight between the two treatment groups was observed. These observations were made by the surgeon who was blinded to treatment allocation and who performed all procedures, as well as noted by a surgeon not involved with the study.

Treprostinil Administration Attenuates Hepatic Cold Ischemia-Reperfusion Injury and Improves Liver Graft Function

In a healthy liver, hepatic aminotransferase enzymes do not leak from the liver into systemic circulation and serum levels are negligible, i.e. 30–40 IU/L. Therefore, serum concentrations of the hepatic enzymes ALT and AST served as markers of hepatic injury. We found that serum ALT and AST levels in the placebo-treated group reached a peak of 2971 ± 156 and 5168 ± 872 IU/L, respectively, at 24 h post-transplantation. Treatment with treprostinil significantly reduced serum ALT and AST levels to 690 ± 98 and 1133 ± 69 IU/L, respectively. At 48 h post-reperfusion, treprostinil significantly reduced serum ALT and AST levels to less than 80 and 90% of those seen in the placebo-treated group, respectively (Figure 1A and B), similar to previous findings.⁹ The extent of liver graft injury and exposure to study drug during the first 48 h post-OLT is reflected in area under the time-concentration curves (AUC) for ALT and AST. The AUC_0–48 of ALT and AST were more three times higher in the placebo- compared to treprostinil-treated group, indicating that treprostinil-treated animals experienced less hepatic injury than placebo-treated animals (Figure 1C).

Figure 1.

Treprostinil reduces hepatic graft injury. Time course of serum (A) ALT and (B) AST levels at 6, 24, and 48 h after reperfusion, (C) AUC_0–48 h of serum ALT and AST post-OLT (IU*h/L) of donor + recipient treatment, and (D) bilirubin concentration at 1, 3, 6, 24, and 48 h post-OLT. ^∗p < 0.05, ^∗∗p < 0.001, and ^∗∗∗p < 0.001 vs. placebo (n = 5).

Total serum bilirubin served as a marker of hepatic function. Bilirubin peaked at 3-h post-OLT (0.38 ± 0.11 mg/dl) and gradually returned to baseline (0.17 ± 0.06 mg/dl) by 24 h post-OLT in the placebo-treated group (Figure 1D). Alternatively, treprostinil-treated animals had a lower peak at 1 h post-OLT (0.30 ± 0.10 mg/dl) and returned to baseline by 3 h post-OLT. These results suggest that administration of treprostinil maintained biliary excretion of bilirubin and preserved liver function early post-OLT.

Treprostinil Suppresses Pro-inflammatory Gene Expression in Graft Liver

In response to injury, pro-inflammatory cytokines are produced, whereas in the normal liver the mRNA levels of cytokines are very low. We examined messenger RNA (mRNA) expression of pro-inflammatory mediators with the use of early (1 h) post-transplant liver graft tissue by qRT-PCR. Treprostinil reduced cold I/R injury–induced up-regulation of TNF-α (p = 0.05) and IL-6 (p = 0.01) mRNA levels, and up-regulated the anti-inflammatory cytokine IL-10, compared with the placebo-treated group (Table 1). Hepatic IL-6 mRNA expression was also decreased by treprostinil treatment, although the value did not reach statistical significance. These results suggest that treprostinil reduced the pro-inflammatory response in liver graft early post-OLT.

Table 1.

mRNA expressions in liver graft at 1 h post-reperfusion.^a

Group	TNF-α	IFN-γ	IL-1β	IL-6	IL-10
Treprostinil	8.0 ± 1.3	1.2 ± 0.6†	1.9 ± 1.0	59.4 ± 12.9	116.0 ± 3.7
Placebo	14.5 ± 2.1	14.1 ± 3.0	1.9 ± 0.2	81.1 ± 26.7	84.3 ± 36.4

TNF-α, Tumor necrosis factor alpha; IFN-γ, interferon gamma; IL, interleukin.

^†p < 0.05 vs. placebo.

^amRNA levels of each gene are expressed as a fold-increase over NL (set at 1.0).

Treprostinil Improves Cytochrome P450 mRNA Expression in Liver Graft

The acute effects of I/R injury on the mRNA expression of CYP450 enzymes in hepatic tissue were measured at 6 h post-OLT by qRT-PCR. Significant differences between the treprostinil- and placebo-treated groups were detected (Figure 2A). Treprostinil improved CYP3A1, 3A2, 3A18, and 2E1 mRNA expression by 2.1-, 1.8-, 1.7-, and 2-fold that of placebo, respectively. The greatest effect of treprostinil was observed with CYP2C11 levels, which were improved 2.6-fold vs. placebo. The early detection of improved mRNA expression suggests that treprostinil regulates CYP450 expression.

Figure 2.

Treprostinil improves hepatic CYP450 mRNA expression in liver graft. The mRNA expression of CYP450 enzymes in liver graft at (A) 6 and (B) 48 h post-reperfusion from NL, treprostinil- and placebo-treated animals. Data are normalized to GAPDH expression in NL and set to 1.0. Results show fold-increase over NL. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. NL; ^†p < 0.05 vs. placebo (n = 3).

To determine if suppression of CYP450 mRNA persisted in the later period post-OLT, CYP450 mRNA expression was examined at 48 h post-OLT. An improvement in CYP450 mRNA expression by treprostinil continued to at least 48 h post-OLT, vs. placebo-treated group (Figure 2B). Hepatic mRNA expression of CYP3A1 and 3A2 in the treprostinil-treated group was 1.7- and 1.6-fold greater than in the placebo-treated group. Treprostinil improved CYP2E1 levels to 49% of NL, vs. 29% of NL in the placebo-treated group. Taken together, these results suggest that treprostinil modulates transcriptional regulation of hepatic CYP450 phase I enzymes.

Treprostinil Restores Cytochrome P450 Protein Expression in Liver Graft

Liver graft microsomes were used to study effects of treprostinil on the protein expression of the three major CYP450 enzymes, CYP3A2, CYP2C11, and CYP2E1, at 48 h post-OLT by Western immunoblotting. Rat CYP3A1 and CYP3A2 share 89% homology¹⁶ and treprostinil significantly improved CYP3A2 protein levels by 2.3-fold that of placebo (Figure 3A). The most notable effect of treprostinil occurred with CYP2E1, where treprostinil restored CYP2E1 protein expression to 100%, vs. 64% of normal in the placebo-treated group (Figure 3B). Protein expression of CYP2C11 was also improved by treprostinil, compared to the placebo group (Figure 3C). These results indicate that the extent of I/R injury on CYP450 protein expression persisted at least up to 48 h post-OLT and treprostinil improved protein expression of the three major CYP450 enzymes in rat liver graft post-transplantation.

Figure 3.

Treprostinil restores hepatic CYP450 protein expression in liver graft. Hepatic microsomal protein (25 μg) was assayed for (A) CYP3A2, (B) CYP2E1, and (C) CYP2C11 from NL (lanes 1–2), treprostinil- (lanes 3–5) and placebo-treated (lanes 6–8) animals at 48 h post-OLT. Densitometry was calculated using Image J software (NIH, Bethesda, MD); data are normalized to GAPDH expression. ^†p < 0.05 vs. placebo (n = 3).

Treprostinil Improves Microsomal Cytochrome P450 Activity in Liver Graft

CYP450 activity is an important indicator of liver graft function in vivo.^1–3,17 Liver graft microsomes were used to study the effects of treprostinil on CYP3A, CYP2E1, and CYP2C11 activity at 1, 3, and 48 h post-OLT. CYP3A activity was determined using the formation rate of 1-OH MDZ from MDZ and 6β-OH TST from TST as substrates. The formation rate of 1-OH MDZ in normal liver (5.5 ± 0.2 pmol/min/mg) was significantly reduced to 50% early post-OLT in both treprostinil- and placebo-treated animals (Figure 4A) at 48 h post-OLT. CYP3A activity in treprostinil-treated animals was 3-fold greater than placebo-treated animals (18% of NL). Similarly, 6β-OH TST formation rates in treprostinil-treated animals were 2-fold greater than placebo-treated animals (Figure 4B).

Figure 4.

Treprostinil improves CYP450 activity in liver graft. Formation rates of (A) 1-hydroxymidazolam, (B) 6β-hydroxytestosterone, (C) 6-hydroxychlorzoxazone, (D) 2α-hydroxytestosterone, and (E) 16α-hydroxytestosterone in hepatic microsomes from normal liver (NL), treprostinil- and placebo-treated animals at 48 h post-OLT. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. NL; ^†p < 0.05 vs. placebo (n = 3).

The activity of CYP2E1 was determined using the formation rate of 6-OH from CZN. At 48 h post-transplantation, treprostinil significantly increased CYP2E1 activity by more than 2-fold (73% of NL) of placebo-treated animals (Figure 4C).

The major component of microsomal CYP450 in male rat liver is CYP2C11.¹⁸ The formation rates of 2α- and 16α-hydroxytestosterone from testosterone served as substrates for CYP2C11 activity. The 2α- and 16α-hydroxylation of testosterone markedly decreased in the placebo-treated group to 16% and 25% of normal, respectively, at 48 h post-OLT (Figure 4D and E, respectively). In contrast, treprostinil improved 2α- and 16α-hydroxylation of testosterone to 56% and 40% of normal, respectively. These results suggest that cold I/R injury significantly reduced the CYP450 activity early post-OLT and that treatment with treprostinil significantly improved CYP450 activity in the liver graft post-OLT.

Discussion

The current study demonstrates that treprostinil, a PGI₂ analog, significantly improves CYP3A, 2E1, and 2C11 expression and/or activity in liver graft tissue following cold I/R injury in a clinically relevant rat orthotopic liver transplantation model. Several studies have investigated whether the activity of hepatic CYP450 enzymes in experimental models of liver inflammation or infection are down-regulated, which can cause dose-dependent drug toxicity associated with impaired drug clearance in vivo.^19,20 Yet, data that directly examines effects of cold I/R injury on CYP450 activity during OLT has not been obtained. Knowing the extent of cold I/R-induced injury on hepatic CYP450 activity prior to liver transplantation can serve as a guide in proper drug therapy, especially in the early post-operative period, and increase the number of available organs for transplantation.

During the host response to inflammation, inflammatory mediators, including release of pro-inflammatory cytokines, have been associated with altered content, expression, and activity of CYP450 enzymes, consequently leading to alterations in the metabolism and elimination of certain drugs.⁶ The losses in drug metabolism are channeled predominantly through the production of cytokines, which modify the expression and function of specific transcription factors that regulate CYP450 gene expression, i.e. Nuclear Factor-Kappa B (NF-κB). Several studies have demonstrated that the activities of many hepatic CYP450 enzymes in experimental models of liver inflammation or infection and in man are down-regulated, which can cause dose-dependent drug toxicity associated with impaired in vivo drug clearance.^19,20 In most cases, the decreased activity is accompanied or preceded by decreased hepatic levels of the corresponding CYP450 mRNA and protein expression.⁶ Although, the different models of inflammation can result in different rates of drug clearance and or reduced microsomal metabolism of drugs.²¹ There is evidence for both transcriptional and post-transcriptional down-regulation of CYP450 mRNA by inflammatory stimuli.²² For example, the down-regulation of CYP2C11 following treatment with bacterial LPS or other inflammatory responses has been shown to primarily occur via decreased mRNA expression, which is followed by a similar decrease in protein levels.^6,23,24 The CYP2C11 promoter contains an NF-kB binding site and mutation of the promoter prevented IL-1-mediated CYP2C11 suppression.¹⁸ Other proposed mechanisms that apply to specific CYP450 enzymes involve post-translational modifications or increased degradation.²⁵ CYP2E1 has been shown to be most affected by inflammation at the protein level,^26,27 through mechanisms to prevent degradation, i.e. protein stabilization.^28–31 In the current study, the finding that CYP2E1 protein expression was restored to normal, suggests that CYP2E1 protein is stabilized or its degradation was prevented by treprostinil. Additional studies are required to elucidate the mechanisms involved. Also, the different patterns of CYP2C11 mRNA expression and activity support the idea that different inflammatory mediators regulate CYP450 expression and are enzyme-specific. Inhibition of pro-inflammatory cytokines is a major pathway responsible for the improved hepatic CYP450 expression and activity in the treprostinil-treated group.

Administration of LPS is a classic model of bacterial sepsis, however, different concentrations of LPS and cytokines administered in vivo or in vitro can have enzyme-selective effects on CYP450 expression.^6,32 In a model of cold graft storage followed by reperfusion using a recirculation method, Izuishi et al examined the effects of prolonged cold graft storage on CYP450 content, protein and activity. Significant changes were observed only after 48 h of cold storage, which is not clinically feasible.⁴ In a rat model of partial ischemia (70%), 1 h of warm ischemia followed by 3 h reperfusion resulted in no significant changes in CYP2E1 or CYP2C11 protein, while CYP3A2, CYP2E1, and CYP2C11, activity decreased by 30%, 17%, and 34%.^5,33–34 In a porcine model of warm ischemia, after 6 h of partial hepatic occlusion, the activities of CYP3A, CYP2E1, and CYP2C were decreased to 31%, 62%, and 62%, respectively, while CYP3A protein expression remained unchanged.³⁵ Recently, Aguilar-Melero et al showed reduced liver I/R injury by cardiotropin-1 administration to donor pigs.³⁶ Considering the various models used in these studies, it is difficult to translate the results from a model of warm partial I/R injury or a liver graft reperfused ex vivo to the clinical setting, which involves cold ischemia of a finite time period and followed by warm reperfusion, thereby invoking different cellular injuries and, consequently, different patterns of CYP450 expression in the host response. The patterns of mRNA expression, protein levels and CYP450 activity in the present study support the theory that different inflammatory mediators regulate CYP450 expression at different levels and are enzyme-specific, depending on source of injury.

Central to the mechanism of cold I/R-associated liver injury is the activation of the pro-inflammatory cascade resulting in the release of pro-inflammatory cytokines. In agreement with the present study, serum and hepatic mRNA levels of TNF-α, IL-6, and ICAM-1 were all significantly up-regulated early post-OLT following 18 h cold liver graft storage.^37,38 PGI₂ analogs have been shown to inhibit leukocyte activation by inhibiting TNF-α production, neutrophil activation and adhesion to endothelial cells^39,40 and, in particular, treprostinil inhibited the mRNA expression of multiple cytokines including TNF-α, IL-6, and IL-1β by blocking the translocation of NF-kB.⁴¹ In a separate study, we measured CYP450 induction in primary cultured hepatocytes and found that treprostinil did not induce CYP450 mRNA, thereby eliminating the possibility that treprostinil improved CYP450 activity by enzyme induction (Ghonem et al, in review).

Considering the many factors involved in I/R injury and the role of PGs in maintaining vascular and cellular homeostasis, PGI₂ has a particular relevance in the setting of hepatic I/R injury associated with OLT. Since the late 1980s, PG analogs have been tested for their ability to reduce I/R-induced liver injury in several animal models and in clinical liver transplantation. Early studies using epoprostenol and iloprost were promising in minimizing primary liver graft non-function, however, clinical application has been limited by their inherent instability and short half-life, thus requiring intolerable doses. While the principle pharmacological effects of PG analogs are similar, there are notable differences in the pharmacokinetics and metabolism, with a wide range in half-lives. Treprostinil has favorable characteristics, including longer stability in the delivery system (48 h at room temperature) and a longer half-life, which enables lower doses to achieve therapeutic efficacy with lower potential for side effects.

The current study demonstrated that I/R injury post-OLT resulted in a significant decrease in hepatic CYP450 activity, secondary to increased pro-inflammatory cytokines in liver graft post-OLT. Noteworthy findings are that treprostinil protected the liver graft against I/R injury, suppressed the pro-inflammatory response, and improved CYP450-mediated drug metabolism. From the clinical perspective, donor + recipient treatment may not always be possible. Therefore, to determine whether recipient only treatment yields protection, we treated recipient animals with treprostinil or matching placebo (100 ng/kg/min) prior to transplantation and until the time of sacrifice.⁹ The significant reduction in serum ALT and AST levels post-OLT confirms treprostinil as a viable therapy to protect patients against I/R injury during OLT (Supplemental Material 3).

In summary, this study demonstrates for the first time that treprostinil significantly improves the expression and activity of the major hepatic CYP450 enzymes in rat liver graft early post-OLT. Our findings suggest that treprostinil improves liver graft function early post-OLT. Treprostinil has the potential to serve as a therapeutic option to protect the liver graft against cold I/R injury during OLT and increase the number of suitable organs available for liver transplantation.

Conflicts of interest

All authors have none to declare.

Appendix A. Supplementary material

The following is/are the supplementary data related to this article: