*4.1. Endogenous Toxic Substances*

Ammonia is an endogenous metabolite produced primarily from the breakdown of amino acids. It is taken up by the liver and converted in the urea cycle, but accumulates in inborn errors of metabolism and hepatic failure. The accumulation of ammonia in hepatic failure is pathogenic in the cerebral oedema seen with this condition. Ammonia is a lipid-soluble weak base and, as such, represents a rational target for entrapment by pHgradient liposomes with an acidic centre. Forster et al. used LSPD to entrap ammonia in rat models with greater efficacy than conventional PD [11]. Agostoni et al. used pH-gradient liposomes in LSPD to treat hyperammonemia in rat models of hepatic cirrhosis [14]. This technique resulted in a 10-fold increase in dialysate ammonia removal compared with conventional PD and reduced both ammonia concentrations in the plasma and the degree of brain oedema. Liposomes in this experiment were prepared using an osmotic shock technique, whereby liposomes suspended in water were "shocked" by the sudden addition of citric acid to achieve pH 2. The sudden osmotic change induced transient increased permeability in the liposome membrane with subsequent pH equilibration. The suspensate was then neutralised with an alkaline xylitol-based solution. Preparation of liposomes in

this way could markedly increase the storage life, with pH neutralisation as the last step prior to use. Giacalone et al. demonstrated in 2018 that pH-gradient liposomes maintained their ability to sequester ammonia in vitro in ascitic fluid from patients with liver disease when co-incubated with drugs commonly administered to this patient group such as betablockers and diuretics [15]. In the same study it was demonstrated that LSPD did not remove more essential endogenous metabolites than conventional PD fluid.

Matoori et al. investigated the pharmacokinetics and safety profile of LSPD using pH-gradient liposomes with acidic centres in dialysate in healthy minipigs [16]. This study was undertaken with a view toward meeting safety requirements for approval for a first-in-human study. Two doses of liposomes were administered intraperitoneally daily for ten days to healthy minipigs, as well as in a pig model of hyperammonaemia. The results demonstrated the pharmacokinetics of citric acid (the driver of the liposome core pH gradient) to be linear and the minipigs showed only low systemic accumulation of phospholipids. In addition, there were no complement-activation-related pseudoallergy reactions and the LSPD was tolerated well. Finally, in a hyperammonaemic pig model, pH-gradient liposome-containing dialysate was found to have significantly higher levels of ammonia in the peritoneal fluid when compared with the liposome-free control group [16]. Given that hyperammonemia carries a poor prognosis in liver failure, with the risk of encephalopathy, coma and death, and that there are limited treatment strategies available, LSPD represents a promising treatment avenue. The website of the patent-holders for this technology lists their pH-gradient with acidic centre liposomes in dialysate as being at the phase 1 stage of preclinical development.
