The exchange of lipids with cells and other lipoproteins is a crucial process in HDL metabolism and for HDL antiatherogenic function. Here, we tested a practical method to quantify the simultaneous transfer to HDL of phospholipids, free-cholesterol, esterified cholesterol and triacylglycerols and to verify the lipid transfer in patients with coronary artery disease (CAD) or undergoing statin treatment. Twenty-eight control subjects without CAD, 27 with CAD and 25 CAD patients under simvastatin treatment were studied. Plasma samples were incubated with a donor nanoemulsion prepared by ultrasonication of the constituent lipids and labeled with radioactive lipids; % lipids transferred to HDL were quantified in the HDL-containing supernatant after chemical precipitation of non-HDL fractions and the nanoemulsion. The assay was precise and reproducible. Increase of temperature (4-37 degrees C), of incubation period (5 min to 2 h), of HDL-cholesterol concentration (33-244 mg/dL) and of mass of nanoemulsion lipids (0.075-0.3 mg/microL) resulted in increased lipid transfer from the nanoemulsion to HDL. In contrast, increasing pH (6.5-8.5) and albumin concentration (3.5-7.0 g/dL) did not affect lipid transfer. There was no difference between CAD and control non-CAD with regard to the lipid transfer, but statin treatment reduced the transfer to HDL of all four lipids. The test herein described is a valid and practical tool for exploring an important aspect of HDL metabolism.

A cholesterol-rich microemulsion (LDE) that binds to low-density lipoprotein (LDL) receptors is selectively taken up by malignant cells that overexpress those receptors and may be used as vehicle for antineoplastic agents. This study aimed to develop the association of etoposide with LDE. It was firstly observed that etoposide poorly associates with the microemulsion, therefore the experiments were performed with a lipophilic fatty acid derivative of the drug. The association of etoposide oleate with LDE was almost 100% and was tested for physical and chemical stability, as well as for cellular uptake, toxicity in mice and cytotoxic activity against a neoplastic cell line (NCI-H292). Uptake and cytotoxic activity of LDE-etoposide oleate by NCI-H292 cells was mediated by LDL receptors. The anti-proliferative activity of LDE-etoposide oleate against the neoplastic cells was smaller than that of etoposide oleate (IC50 (drug concentration required to inhibit 50% of the cell growth) = 0.48 and 0.19 mM, respectively). This difference, however, can be ascribed to the activity of the commercially used vehicle and not the drug itself because when this vehicle was added to the cultures with LDE-etoposide oleate, the IC50 decreased. On the other hand, the tolerability of LDE-etoposide oleate to mice was remarkable, such that its lethal dose (LD50) was about five-fold that of the commercial formulation (LD50 = 315 and 58 mg kg(-1), respectively). In conclusion, LDE-etoposide oleate association is stable and the cytostatic activity of the drug is preserved while its toxicity to animals is small. By diminishing the side effects and directing etoposide to neoplastic tissues, LDE may be regarded as an advance in chemotherapy with this drug.

A cholesterol-rich nanoemulsion (LDE) may be used as a vehicle to target antineoplastic drugs against cancer cells. The association of an etoposide derivative to LDE is stable and retains the cytotoxic activity of etoposide. We have evaluated the toxicity and antitumoral action of this new preparation in-vivo. Melanoma-bearing mice and control mice were administered LDE-etoposide oleate or commercial etoposide, either with or without radioactive labelling. The maximum tolerated dose (MTD), tissue distribution, plasma decay curves, pharmacokinetic parameters and antitumoral activity were determined. Association to LDE drastically reduced the drug toxicity, since MTD was approximately five-fold greater than in commercial etoposide. LDE-etoposide oleate was concentrated four-fold in the tumour compared with the normal adjacent tissues, was removed faster from plasma in tumour-bearing mice than in controls, and remained in the bloodstream longer than commercial etoposide. The tumour growth inhibition rate and survival were greater in animals treated with LDE-etoposide oleate compared with commercial etoposide. However, increasing the dose from 17 to 85 M kg À1 did not result in further improvement of the antitumour action. The incorporation of etoposide oleate to LDE resulted in markedly reduced toxicity and superior antitumoral activity. LDE-etoposide oleate is a promising new weapon for cancer treatment.

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