Caspase inhibition in liver transplantation: from basic research to clinical studies

During transplantation, the liver is subjected to ischaemia and reperfusion (I-R) injury at three distinct stages. The first, when the liver is explanted from the donor and stored on ice at 0° to 4°C, is a variable but generally long period of cold ischaemia. The time of anastomosis, when the liver is removed from ice until its implantation in the recipient, represents the second, relatively shorter stage of I-R injury. In this period of ischaemia, the liver warms slowly up towards body temperature at a rate of 0.5°C per minute. Normothermic reperfusion of the implanted liver with the recipient's blood at 37°C delineates the third stage. This cold ischemia/normothermic reperfusion injury causes up to 10% of early liver transplant failures and can lead to initial poor graft function (IPGF), acute and chronic rejection.¹ The pathogenesis of cold ischaemia/normothermic reperfusion injury represents a complex interplay between biochemical, cellular, endothelial and tissue specific factors, with inflammation being a common feature.

Liver cells seem to die mainly by necrosis. While cold storage appears to cause injury predominantly to non-parenchymal cells (Kupffer, sinusoidal endothelial and stellate cells), normothermic ischaemia injury affects above all parenchymal cells such as hepatocytes.² The complement system plays an important role in the development of cold ischaemia/normothermic reperfusion injury as a manifestation on innate immunity. During cold storage, complement fragment 4d deposition and neutrophil infiltration were found in lobar and periportal necrotic hepatocytes resulting in IPGF of the liver after transplantation.³

However, liver cell death during cold ischaemia/normothermic reperfusion is not only the result of necrosis but, also of apoptosis or programmed cell death.⁴ These different mechanisms of cell death occur simultaneously during cold ischaemia, and they even imbricate during reperfusion. Necrosis and apoptosis are interdependent phenomena resulting from activation of shared pathways and signals.⁵

The apoptotic process is executed actively by cysteinyl-aspartate-specific proteases, the caspases, and occurs in a programmed fashion.⁶ Synthesized as inactive precursors, caspases are activated upon specific cleavage at defined aspartate residues. Caspase activation can be triggered by an extrinsic molecular pathway mediated by death receptors (Fas, TNFR1 and TRAIL) on the cell surface and by an intrinsic molecular pathway, induced at the mitochondrial level.⁶ In this apoptotic process, the ‘initiator’ caspases-8, -9 and -10 cleave and activate ‘executioner’ caspases, mainly caspase-3, -6 and -7. The caspase activation results in morphological cellular changes such as shrinkage of the cell, condensation of chromatin and disintegration of the cell into small apoptotic bodies. Finally, these apoptotic bodies are phagocytosed mainly by Kupffer cells without inflammatory reaction. Therefore, the caspase pathway plays an important role in clearing apoptotic dead cells in a way that allows remodelling of the liver cell plates without inflammation. However, this tissue remodelling is dependent not only on the degree of mitosis and apoptosis but, also on the balance between cell death and cell clearance and a mismatch between the execution phase of apoptosis and cell disposal may contribute to cold/normothermic reperfusion injury of the liver.

In liver transplantation, the extent of liver injury seems to be mirrored by caspase activity. Indeed, a set of genes involved in the regulation of apoptosis were highly expressed in transplanted livers of patients with IPGF.⁷ In these livers, an increase in caspase-3 activity was also detected.⁷ During transplantation, the initial liver graft damage occurs during cold preservation, but at the time of reperfusion, the rapid metabolism of accumulated metabolic products leads to the production of toxic molecules, for example free radicals, and to the activation of apoptosis. Ischaemia by itself can trigger apoptosis, but reperfusion accelerates the process. Using fluorescence microscopy, caspase activity has been identified particularly in cells localized in the perivascular areas of the reperfused liver.⁸

Pharmacological intervention applied before and/or at the onset of reperfusion can reduce cell death. Several animal studies have been performed to test a variety of inhibitors targeting caspase-mediated liver cell apoptosis. Initial studies⁹ examined the effects of a pan-caspase inhibitor Z-Asp-2,3-dichlorobenzoyl-oxymethylketone (Z-Asp-cmk), in a rat normothermic liver I-R model. Treatment with Z-Asp-cmk was associated with lower aminotransferase serum levels, reduced caspase-3 activity, reduced apoptosis and improved survival. These experimental data have paved the way for testing several caspase inhibitors in animals to reduce liver apoptosis and consequently liver I-R injury during liver transplantation.^10,11 For example, IDN-6556, a novel irreversible broad-spectrum caspase inhibitor, has been shown to reduce cold ischaemia/normothermic reperfusion-induced apoptosis and injury of the liver.^10,11 Collectively these studies have provided solid preclinical evidence that the inhibition of liver cell apoptosis attenuates cold ischaemia/normothermic reperfusion injury and potentially improves the quality of liver grafts. However, inhibition of the caspase pathway may be detrimental by blocking non-inflammatory apoptotic cell clearance and thereby promoting inflammation. The use of the pan-caspase inhibitor z-VAD-fmk may induce macrophage overexpression and secretion of several chemokines and cytokines, including TNFα, which results in tissue inflammation prior to the death of these macrophages by necrosis.¹² Furthermore, when apoptotic cell death is blocked by z-VAD-fmk, some cells will undergo an alternate form of cell death that has distinctive characteristics and is called necroptosis, a form of programmed cell death with characteristics of necrosis.¹³ Finally the same pan-caspase inhibitor, by blocking caspase activation, may induce autophagic cell death (self-digestion).¹⁴

As caspases seem to be key mediators of apoptosis, immune response, complement activation and inflammation pathways, inhibiting one of these pathways individually is probably not sufficient to prevent liver damage after cold ischaemia/normothermic reperfusion. In contrast, blockade of caspases, which represent the point of convergence of these different pathways, could offer an interesting strategy for ameliorating liver injury, via modulation of inflammation, immune response and apoptosis.¹⁵

A phase I clinical trial of the pan-caspase inhibitor IDN-6556 administered to patients with hepatic impairment showed that the drug was well tolerated and contributed to a marked reduction in serum aminotransferase levels¹⁶. These results were confirmed by a recent study in which oral IDN-6556 lowered aminotransferase serum activity in patients with chronic hepatitis C.¹⁷ In clinical practice, administration of drugs could be easily performed during graft preservation before its implantation in the recipient and/or before graft revascularization and reperfusion. Pan-caspase inibitor IDN-6556 has a short half-life (1-h half-life) and this is very important, because long-term antiapoptotic effects may have unintended consequences.¹¹ Adding the drug to the standard preservation solution seems ideal because the time of exposure is limited and the substance is likewise washed out prior to revascularization. Since then, the pan-caspase inhibitor IDN-6556 was successfully tested in a Phase II clinical trial in human liver transplantation.¹⁸ The IDN-6556 was administered in cold storage and flush solutions during liver transplantation and provided local therapeutic protection against cold ischaemia/normothermic reperfusion-mediated apoptosis and injury. There was no delayed graft function, no primary non-function and no retransplantation. So, despite the limited number of patients in this study, a further step seems to have been achieved in liver graft protection by caspase inhibitors.¹⁸

The improved knowledge of apoptotic mechanisms during cold ischaemia/normothermic reperfusion injury and the possibility to inhibit the activation of the caspases involved in this type of injury by caspase inhibitors derived from animal studies and its translation to clinical trials highlights the need and the importance of basic research in liver transplantation.

Conflicts of interest

None declared