Therapeutics administered during ex vivo liver machine perfusion: An overview

Table 1 Major categories of ex vivo machine perfusion therapeutics in liver transplantation

Category	Agents
RNAi pathway	siRNA
	Anti-sense oligonucleotide (Miravirsen)
Defatting cocktail	Variable
Vasodilators	Prostacyclin
	BQ123 and Verapamil
	Prostaglandin E1
Others	Anti-inflammatory agents
	Human liver stem cells extracellular vesicles
	δ-opioid agonist (Enkephalin)
	NLRP3 inflammasome inhibitor mcc950

RNAi: Ribonucleic acid interference; siRNA: Small interfering ribonucleic acid; NLRP3: Nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3.

Table 2 Other major therapeutic additives in models of ex vivo liver machine perfusion

Ref.	Ex vivoperfusion type	Therapeutic	Problem	Animal	Model	Samplesize	Ex vivo perfusiontime (h)	Outcomes
Goldaracena et al[9], 2016	SNMP	Anti-inflammatory agents	IRI	Pig	Transplantation	5	4	During EVLP: lower AST, TNF-α, IL-6
								Lower HA levels, β-galactosidase and higher IL-10 (nonsignificant)
								After transplantation: Lower bilirubin, lower IL-6, lower cleaved caspase 3 staining, intact sinusoidal endothelial cell lining
		(Alprostadil, n-acetylcysteine, carbon monoxide, sevoflurane)
								Lower AST, TNF-α, HA,
								ALP and higher IL-10 (nonsignificant)
Rigo et al[65], 2018	NMP	HLSC-EV	IRI	Rat	EVLP	9	4	HLSC-EV uptake in treated livers
								Reduced necrosis and apoptosis on histology, lower Suzuki tissue injury score, lower apoptosis, lower AST and LDH, lower HIF-1α & TGF-β1 (hypoxia induced markers)
								NMP had low hematocrit to induce hypoxia
Beal et al[66], 2019	NMP	Enkephalin	IRI	Rat	EVLP	6	4	10 μmol/L determined to be optimal concentration in an in vitro model: Lower ALT and MDA; better preservation of structural architecture; decreased caspase-3 expression;
		(δ-opioid agonist)
								decreased TUNEL staining; decreased phosphorylation of p38 and JNK; increased expression of p-Akt, PI3K, p-Bad and Bcl-2
Yu et al[67], 2019	HMP	NLRP3 Inflammasome	IRI	Pig	Transplantation	6	2	All reduced in HMP-postop group with added mcc950 in perfusate and IV administration of mcc950
								After transplantation: TNF-α, IL-1β, β-galactosidase, post-reperfusion serum ALT and AST, MDA, apoptosis staining, caspase-1 levels
		Inhibitor mcc950

SNMP: Subnormothermic machine perfusion; NMP: Normothermic machine perfusion; HMP: Hypothermic machine perfusion; IRI: Ischemia-reperfusion injury; EVLP: Ex vivo liver perfusion; AST: Aspartate aminotransferase; HA: Hyaluronic acid; ALP: Alkaline phosphatase; HLSC-EV: Human liver stem cells-derived extracellular vesicles; LDH: Lactate dehydrogenase; ALT: Alanine aminotransferase; MDA: Malondialdehyde; IV: Intravenous.

Table 3 Summary of proposed advantages and limitations of liver machine perfusion therapeutics

Therapeutic	Advantages	Limitations/future considerations
RNAi pathway	Selectively targets and silences/degrades specific genes	Most beneficial/effective siRNA target against liver IRI in transplantation remains to be identified
	Mechanism of siRNA silencing pathway is generally understood	Potential for administration of multiple siRNA constructs each with a different target
	Organ-specific uptake	Requires design of siRNA against target mRNA and validation of target silencing
	Permits imaging studies visualizing tissue uptake and distribution
Defatting cocktails	Ability to restore liver function by defatting	Steatotic livers are already predisposed to IRI[32]
	Aimed at enhancing natural lipid metabolism via lipid export, reduction of triglyceride levels, and stimulation of lipid oxidation and ketogenesis	Mechanisms of glucose and lipid control in liver remain poorly defined[69]
		Undefined consensus for quantifying degree of steatosis[70]
		Need for perfusate exchange protocol as secreted triglycerides recirculate causing further increase in lipid deposition[47]
		Kinetics of defatting may surpass average timeframe of liver transplantation[48]
Vasodilators	Focused on improving intrinsic function of liver to improve blood flow via smooth muscle relaxation and vasodilation	Effects of vasodilators in marginal grafts remains unclear[61]
	Act to increase arterial flow and decrease post-sinusoidal resistance[71]	Combination of agents does not allow for specific identification of most beneficial agent(s)
Anti-inflammatory agents	Some agents also act as vasodilators[68,72,73]	Mechanisms of anti-inflammatory agents remains unexplored in context of ex vivo liver perfusion[9]
	Act to scavenge free radicals to prevent IRI[74]	Combination of agents does not allow for determination of which specific agents were beneficial[9]
	Ability to protect other cell types such as endothelial cells[75,76]
HLSC-EV	Regenerative and hepatoprotective properties[77,78]	Unknown mechanism of hypoxic protection[65]
	Diverse differentiating capabilities[77]	Timing of EV uptake during NMP currently unknown[65]
	Contain mRNA and miRNA subsets that modulate activity of target cells[79]
	May serve as option for liver diseases without need for stem cells transplantation[65]
δ-opioid agonist (Enkephalin)	Protects against oxidative stress[66]	Unknown therapeutic role in setting of post-perfusion liver transplant model with measured outcomes of graft function[66]
	Prevention of mitochondrial dysfunction via opioid receptor signaling[66]
	Protection against IRI by slowing cellular metabolism[80,81]	Unknown role in cold ischemia conditions for liver transplant models[66]
NLRP3 inflammasome inhibitor (mcc950)	Blocks NLRP3-inflammsome activation preventing inflammatory liver damage[82,83]	mcc950 half-life is 3.27 h, while NLRP3 inflammasome activation lasts for several days after reperfusion[84,85]
	Reduces apoptosis post liver transplantation[67]	Additional mcc950 inhibition studies involving in vitro and in vivo models needed[67]

HLSC-EV: Human liver stem cells extracellular vesicles; NLRP3: Nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3.