Studies On Intestinal Lymphatic Transport Of Fenofibrate From Its Lipid-based Delivery System

Fenofibrate (FEF), a third-generation synthetic phenoxy-isobutyric acid derivative, is particularly effective in reducing levels of triglyceride (TG) and increasing levels of high-density lipoprotein-cholesterol (HDL-C) in patients with dyslipidemias. Compared with an HMG-CoA reductase inhibitor (statin), greater improvements in lipid levels (characterised by high TG levels) are seen when FEF is administered. In recent studies, FEF has also shown many nonlipid effects (e.g. the efficacy with regards to reducing and preventing progression of macrovascular and microvascular diseases in patients with metabolic syndrome or type 2 diabetes mellitus) In addition, most recently, it was found that FEF exerts neuroprotective effects in traumatic brain injury, regulates retinal endothelial cell survival and potently suppresses primary tumor growth in mice, which indicate that FEF may have novel therapeutic properties.FEF is a Biopharmaceutical Classication System (BCS) ClassⅡdrug with a high dose number.The drug is hydrophobic (log P 5.24), with TG solubility>50 g·L-1. In aqueous medium, FEF exhibits very poor solubility (of less than 0.2912μg·ml-1 at 37℃). FEF is a prodrug that is converted into the pharmacologically active metabolite fenofibric acid (FEFA) by esterases when administered orally. The oral bioavailability of FEF is low when taken orally on an empty stomach, even the newest marketed formulations, micronized FEF 200 mg capsules and microcoated FEF 160 mg tablets, require administration with meals to increase bioavailability. Food, especially high-fat food has significantly improved the bioavailability of FEF. The precise mechanisms of this phenomenon have not been well elucidated. Generally, a high-fat meal, coadministrated with a drug, may enhance drug uptake via the portal system delivered directly to the liver by increasing the drug solubility and membrane permeability. Although this may be the most conventional way, other ways also exist and deserve consideration. A high-fat meal can stimulate the production of chylomicrons (CM), which promote the intestinal lymphatic transport of some lipophilic drugs. Those drugs may enter the intestinal lymphatic system in association with lipoproteins within the enterocyte which are the triglyceride core of CM. The increased total systemic bioavailability of FEF in high-fat fed conditions may be related to the promotion of intestinal lymphatic absorption. Thus, we carried out a study on intestinal lymphatic transport of fenofibrate from its lipid-based delivery system.The paper mainly focused on investigating the intestinal lymphatic transport of FEF in rats. Firstly, the uptake of FEF by plasma derived isolated chylomicrons ex vivo was studied in order to predict the lymphatic bioavailability potential of FEF. Secondly, to develop an appropriate animal model for investigating the processes involved in the uptake of lipid-based formulations of FEF via the intestinal lymphatic system, two types of rat models were established and compared. The study was divided into three parts, detailed as follows:PartⅠ, since the simultaneous determination of FEF and FEFA in vivo has not been reported before, a specific and accurate reversed-phase HPLC-UV method was developed and fully validated for simultaneous determination of FEF in rats’plasma, and FEFA and FEF in rats’lymph fluid. Plasma and lymph fluid samples were pretreated by acetonitrile:1M hydrochloric acid (95:5, v/v) protein precipitant containing internal standard naproxen and then analysed by the validated HPLC method. The analytical column was a Calesil ODS (4.6 mm×150 mm,5μm). The mobile phase was composed of methanol and 0.4% phosphoric acid solution (pH 3.2) (82:18, v/v). The flow rate was 1.0 mL·min-1. The wavelength of UV-detector was set at 286 nm. The typical retention time of FEFA, FEF and IS were 4.25,9.15 and 3.3 min, respectively. The calibration curves showed good linear regression in the range of 0.2～50μg-mL-1 (r>0.999) for plasma and lymph fluid samples, intra-and inter-day variations were less than 9%, accuracy was 91%～105%, and extraction recovery was more than 80%. Samples were stable for at least three freeze-thaw cycles, for at least 24 h after being extracted and for at least 12 h and 2 months at room temperature and -30℃, respectively. In conclusion, the established and validated RP-HPLC-UV method is simple, sensitive and accurate, and can be used for pharmacokinetics studies of FEF in rats.PartⅡ, the degree of uptake of FEF by chylomicrons ex vivo was studied, and the difference of intestinal lymphatic transport of FEF in anesthetized and conscious rat models were compared after intraduodenal administration of a single dose of 30 mg·kg-1 of FEF suspension. As a result, the association of FEF with plasma derived isolated chylomicrons is 7.43%±0.6%, which indicated that the absorption of FEF may via intestinal lymphatic transport. The total volumes of lymph fluid within 12 h were (15.18±0.84, n=3) mL and (8.52±0.30, n=3) mL, and the intestinal lymphatic transport of FEF within 12 h were (0.56%±0.03%, n=3) and (0.42%±0.03%, n=3) for conscious and anesthetized rats, respectively. Lymph flow rate was significantly higher (more than twofold) in the conscious animals in comparison to the anesthetized animals at all time point. Thus, the conscious rat model is more suitable for the study of intestinal lymphatic transport of FEF from its lipid-based delivery system when administered orally.PartⅢ, two lipid-based formulations of FEF were prepared and compared with lipid-free micronised FEF and FEF standard suspension with regards to intestinal lymphatic transport. In experimental group, the intestinal mesenteric lymph duct and jugular vein were cannulated in fully conscious, unrestrained rats. In control group, animals were sham operated in terms of the mesenteric lymph duct. A single dose of 30 mg-kg-1 of each FEF formulations was administered orally via gavage in the experimental group (lymph cannulated rats) and the control group (lymph non-cannulated rats). Blood sampling was lasted for 24 h in both groups and lymph collection was lasted for 12 h for model rats additionally. As a result, the cumulative lymphatic transport of self-emulsifying FEF, self-microemulsifying FEF, micro-FEF suspension and FEF standard suspension (expressed as the percentage of dosed FEF appearing in the mesenteric lymph up to 12 h) were 3.52%±0.48%,7.33%±0.81%,2.56%±0.46% and 0.31%±0.06%, respectively. In conclusion, lipid-based FEF formulations versus non-lipid FEF formulations, the former can increase the intestinal lymphatic transport significantly (P<0.05).The objective of this study was to further clarify the mechanism of oral gastrointestinal absorption of FEF and provide some information for the research of FEF new formulations.