Development and characterization of parenteral lipid emulsions from vegetable oil sources to reduce inflammatory responses

Abstract

Lipid emulsions as an integral part of parenteral nutrition provide vital energy to prevent malnutrition when the intake or absorption of nutrients is impaired. Supply of essential fatty acids avoids deficiencies and ensures proper growth and neurological development, which is particularly important in pre-term infants. For many patients, parenteral nutrition is required for life. However, existing formulations suffer from substantial inflammatory adverse effects when used for an extended period. Adverse effects primarily affect the liver and impair its function with cholestasis, steatosis, and fibrosis with potential progression to cirrhosis. Several underlying causes have been proposed, including the oil type and – associated with this – the ratio of n−6 polyunsaturated fatty acids (PUFA) to n−3 PUFA, a high phytosterol content and a low α-tocopherol content. Soybean oil, the most commonly used oil in lipid emulsions, is rich in n−6 PUFAs that are metabolized in the body to pro-inflammatory mediators. The tissue distribution with a high uptake into the liver is thought to further aggravate the observed adverse effects. Evidence has been published that oils rich in n−3 PUFAs cause less inflammatory ad-verse effects and even reverse inflammatory conditions. Available alternative formulations still come with drawbacks, including the non-sustainability due to overfishing of oceans and bear the risk of exposure to lipid-soluble environmental toxins accumulating in the food chain. We aimed at developing intravenous lipid emulsions with a composite oil source rich in n−3 PUFAs achieving a more balanced tissue distribution, ultimately eliciting less adverse effects. Various formulations with different compositions were designed, manufactured and evaluated for improved in vitro and in vivo effects while maintaining compliance with regulatory requirements. Excipients interfering with steps of the lipid oxidation chain reaction were incorporated into the formulation to minimize the extent of oxidative degradation. α-Tocopherol as potent antioxidant also endogenously present in a wide variety of natural oils was added and helped reduce the degree of oxidation to similar levels found in commercially available formulations. Due to the lack of suitable assays for the quantification of degradation products from lipid oxidation and hydrolysis, in-house assays were developed. Three assays were developed or adapted for our needs to characterize the new emulsions with a focus on minimizing sample consumption and achieving rapid sample throughput. Radioactively labelled triglyceride molecules were used to study the in vivo tissue distribution of lipid emulsions produced from different oil sources. Distinct tissue distributions were found based on the used oil type and the n−6 to n−3 balance. At 10 and 60 min after intravenous injection in mice, the highest uptake was seen in lung tissue, followed by the uptake in the liver and spleen. Clearance from blood and plasma was faster for the newly developed n−3 PUFA-rich emulsions than the soybean oil-based emulsions. We also hypothesized that reducing the droplet size would further reduce the uptake in the liver. Several excipients were evaluated for the production of stable emulsions with smaller droplet size. However, reducing the droplet size increased the levels of lipid oxidation products. A compromise between droplet size reduction and oxidation product levels resulted in droplet sizes around 160-170 nm. The smaller droplet sizes did not substantially affect the distribution pattern in vivo as compared to the corresponding emulsions with a droplet size of 280-300 nm, indicating that the oil type is the main driver governing the tissue distribution. Collectively, the feasibility of manufacturing lipid emulsions from a composite oil mixture rich in n−3 PUFA complying with the thresholds set by the United States Pharmacopoeia was demonstrated.