Yolk Phosphatides Research Articles

Effect of oil-in-water lipid emulsions prepared with fish oil or soybean oil on the growth of MCF-7 cells and HepG2 cells

The growth of human breast cancer-derived MCF-7 cells was affected by oil-in-water lipid emulsions prepared with fish oil (FO) rich in n-3 fatty acids (FAs) and egg-yolk phosphatides (EYP) (FO-emulsions), but not by lipid emulsions prepared with soybean oil (SO) and EYP (SO-emulsions). On the other hand, the growth of human hepatocarcinoma HepG2 cells was affected by neither SO-emulsions nor FO-emulsions. The growth inhibition of MCF-7 cells in the presence of FO-emulsions was not affected by trolox, but was inhibited by alpha-lipoic acid, and was even potentiated by ebselen, which works as an antioxidant as well as a lipoxygenase inhibitor. Since prostaglandin E(3), generated from n-3 FAs by cyclooxygenases, has a suppressive effect on tumour cell growth, and increases when lipoxygenases are inhibited, these findings suggest that lipid emulsions incorporating triglycerides of n-3 FAs might be effective in suppressing the growth of MCF-7 cells, possibly via oxidative stress and through eicosanoid production with anti-proliferating activity against cancer cells.

Effect of Oxyethylene Numbers on the Pharmacokinetics of Menatetrenone Incorporated in Oil-in-Water Lipid Emulsions Prepared with Polyoxyethylene-Polyoxypropylene Block Copolymers and Soybean Oil in Rats

We have prepared lipid emulsions of approximately 200 nm in diameter with soybean oil (SO) and a series of Pluronics with various numbers of oxyethylene units and about 60 oxypropylene units (SO/Pluronics), and studied the pharmacokinetics of menatetrenone incorporated into SO/Pluronics in rats. Emulsions of approximately 200 nm in diameter were obtained when SO contents were 2.5% and 20% (w/w) for 2.4% (w/w) PL101 and Pluronics that more than 30% was made up by oxyethylene units, respectively. The half-life of menatetrenone in plasma when oxyethylene units made up less than 30% of the Pluronic (SO/PL101 and SO/PP103) was similar to that for SO/egg yolk phosphatides (SO/EYP), but longer than that when oxyethylene units composed more than 40% of the Pluronic (SO/PP104 and SO/PF108, by 3- and 10-fold, respectively). Pretreatment with dextran sulfate 500000, an inhibitor of emulsion uptake by the reticuloendothelial system (RES), resulted in a higher plasma concentration and a lower liver uptake of menatetrenone as SO/PL101 at 10 min and SO/PP103 at 60 min, indicating that both SO/PL101 and SO/PP103 were taken up by the RES, although SO/PP103 required some time to be recognized by the RES. These findings suggested that larger numbers of oxyethylene units of Pluronics with 60 oxypropylene units were required for the longer plasma circulation of SO/Pluronics due to evasion of the RES.

Effect of acyl chains of phosphatidylcholines on the pharmacokinetics of menatetrenone incorporated in O/W lipid emulsions prepared with phosphatidylcholines and soybean oil in rats.

Oil-in-water (O/W) lipid emulsions were prepared with phosphatidylcholines (PCs) of various acyl chains and soybean oil (SO) using a microfluidizer system, and the pharmacokinetics of menatetrenone incorporated in these oil particles were examined at the clinical injection volume (0.1 mL kg(-1)) in rats. The plasma half-life of menatetrenone incorporated in the oil particles prepared with SO and dipalmitoylphosphatidylcholine (DPPC) (SO/DPPC) was longer than that prepared with SO and eggyolk phosphatides (EYP) (SO/EYP) by 3 fold, while those of menatetrenone as oil particles prepared with SO and either dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) or dilinoleoyl phosphatidylcholine (DLoPC) (SO/DLPC, SO/DMPC, SO/DSPC, SO/DOPC and SO/DLoPC, respectively) were similar to that of menatetrenone as SO/EYP. The menatetrenone uptake by the liver was not significantly different from that as SO/EYP in all SO/PCs examined, but the menatetrenone uptake by the spleen as SO/DPPC and SO/DSPC was higher than that as SO/EYP. The menatetrenone uptake by the lungs as SO/DPPC was also higher than that as SO/EYP. These findings suggest that SO/DPPC is a good candidate drug carrier for the prolonged plasma circulation of lipophilic drugs.

Prolonged circulation of menatetrenone by emulsions with hydrogenated castor oils in rats

Previously, we reported that plasma half-lives of a drug incorporated in lipid emulsions prepared with soybean oil (SO), a long-chain triglyceride, and hydrogenated castor oils (HCOs) (SO/HCOs) were markedly longer, while those as SO/polyoxyethylene sorbitan esters (SO/PSs) were similar, compared to that as SO/egg yolk phosphatides (SO/EYP) [J. Pharm. Pharmacol. 54 (2002) 1357; J. Drug Target. 11 (2003) 37]. In the present study, lipid emulsions were prepared with Miglyol 812 (MO), a medium-chain triglyceride, and HCOs, and the kinetics of the incorporated drug, menatetrenone, were examined. The plasma half-lives and the liver uptake of menatetrenone as MO/polyoxyethylene-(10)-hydrogenated castor oils (MO/HCO10s) were similar to and larger than those as MO/EYP, respectively. On the other hand, the plasma half-lives and liver uptake of menatetrenone as MO/polyoxyethylene-(20)-hydrogenated castor oils (MO/HCO20s) or MO/polyoxyethylene-(60)-hydrogenated castor oils (MO/HCO60s) were markedly longer and lower than those as MO/EYP, respectively. The pretreatment of dextran sulfate 500,000, a reticuloendothelial system suppressor, raised the plasma concentration and inhibited liver uptake of menatetrenone as MO/HCO10, but not for MO/HCO20. These findings suggest that the minimum number of oxyethylene units within HCOs for the prolonged plasma circulation of menatetrenone was 20 for MO/HCOs, similarly to SO/HCOs.

Effect of Oxyethylene Moieties in Hydrogenated Castor Oil on the Pharmacokinetics of Menatetrenone Incorporated in O/W Lipid Emulsions Prepared with Hydrogenated Castor Oil and Soybean Oil in Rats

Lipid emulsions with particle sizes of 190–270 nm were prepared with soybean oil (SO) and a series of hydrogenated castor oils (HCOs) with various oxyethylene numbers, and the effect of oxyethylene numbers of HCOs on the pharmacokinetics of menatetrenone incorporated into the lipid emulsions was studied in rats. Plasma half-life of menatetrenone after administration as the lipid emulsions prepared by HCO with 10 oxyethylene units (SO/HCO10) was similar to that after the administration as SO/egg yolk phosphatides (SO/EYP), but was shorter than that as the lipid emulsions prepared by HCOs with >20 oxyethylene units (SO/HCO20, SO/HCO30, SO/HCO60 and SO/HCO100). Menatetrenone incorporated in SO/HCO10, SO/HCO20 and SO/HCO60 was not taken up by the blood cells in vitro, and the plasma level of menatetrenone incorporated in SO/HCO10 was similar to that of triglycerides, suggesting that menatetrenone was not released from the oil particles even after entering the circulation. Menatetrenone uptake by the liver for SO/HCO10 was similar to that for SO/EYP, while those for SO/HCO20, SO/HCO30, SO/HCO60 and SO/HCO100 was less than that for SO/EYP. These findings clearly demonstrate that 20 oxyethylene units in HCOs is the minimum requirement for the prolongation of the plasma circulation time of menatetrenone incorporated in SO/HCOs.

Effect of Injection Volume on the Pharmacokinetics of Oil Particles and Incorporated Menatetrenone after Intravenous Injection as O/W Lipid Emulsions in Rats

Oil-in-water lipid emulsions are promising drug carriers for lipophilic drugs, however, the pharmacokinetics after entering the circulation should be clarified at clinical injection volume in order to utilize them in a clinical situation. In the present study, the standard lipid emulsions, consisting of soybean oil, egg yolk phosphatides and menatetrenone with diameters of about 150nm, were prepared using a microfluidizer system. The pharmacokinetics of menatetrenone and the oil particles after intravenous injection as standard lipid emulsions at various injection volumes, from the clinical injection volume (0.1 ml/kg) to the experimental injection volume (3.0 ml/kg), were examined in rats.The plasma concentrations of menatetrenone and the oil particles were similar after administration, showing that menatetrenone was not released even after entering the circulation. Menatetrenone was delivered to the liver and spleen at the clinical injection volume, and more menatetrenone was delivered to the liver at clinical injection volume compared with the experimental volume. Moreover, additional information on injection volume-dependency was also obtained from these findings. These results at various injection volumes suggested that the standard lipid emulsions can be utilized as a useful drug delivery system at the clinical injection volume, especially for liver and spleen targeting.

Effect of Composition on Biological Fate of Oil Particles after Intravenous Injection of O/W Lipid Emulsions

Plasma concentrations of oil particles after intravenous injection of oil-in-water (O/W) lipid emulsions were monitored based on the plasma concentration of phospholipids (PL) and triglycerides (TG), and the light scattering intensity (LSI) of plasma. Previously, we found that their time profiles after injection of the standard O/W lipid emulsion composed of soybean oil (SO) and egg yolk phosphatides (EYP) were similar and suggested that the oil particles with diameter of about 200 nm were entrapped by reticuloendothelial system (RES). Herein, in order to develop a delivery system to avoid the RES uptake by using the lipid emulsions, biological fate of lipid emulsions with oil particles of various sizes or those emulsified by surfactants with polyoxyethylene segments were subjected to the investigations.Lipid emulsions with oil particles of various sizes (about 150-550 nm) were prepared by altering EYP content. The oil particles were stable in plasma in vitro, but oil particle size decreased time-dependently after intravenous injection. Plasma clearance of oil particles depended on their initial size and was decreased by pretreatment with dextran sulfate 500 (DS500), a known RES suppressor. These results suggested that oil particles are still entrapped by RES, even for small-sized oil particles (about 150nm). Lipid emulsion with small-sized oil particles was also prepared using medium chain triglycerides. The oil particles were stable in vitro, but the time profiles of plasma concentrations of PL and TG, and LSI of plasma were different, and oil particle size decreased time-dependently after intravenous injection. Plasma clearance of the oil particles also depended on their initial size and was decreased by DS500, suggesting that in vivo instability could be due to RES-mediated processes. Artificial surfactants with polyoxyethylene segments, HCO-60 (HCO60) and polysorbate 80 (PS80), were used for RES avoidance. HCO60 resulted in drastic reduction of the plasma clearance of the oil particles for both lipid emulsions composed of soybean oil and medium chain triglycerides. The time-dependent decrease of oil particle size after intravenous injection was marginal. In contrast, PS80 could not prolong the circulation time of the oil particles, and their size decreased time-dependently after intravenous injection.

O/W Lipid Emulsions for Parenteral Drug Delivery. III. Lipophilicity Necessary for Incorporation in Oil Particles Even After Intravenous Injection

The potential usefulness of oil-in-water (O/W) lipid emulsions as injectable drug delivery systems was examined. Plasma concentrations of oil particles after intravenous injection of a standard lipid emulsion composed of soybean oil and egg yolk phosphatides were monitored based on the plasma concentrations of phospholipids and triglycerides, and the light scattering intensity of the plasma. Their time profiles were similar to each other, and the oil particle size decreased time-dependently. Pretreatment with dextran sulfate, a known reticuloendothelial system (RES) suppressor, resulted in marked reduction of the plasma clearance of the oil particles and of the time-dependent alteration of oil particle size, suggesting that oil particles were trapped by RES. The lipophilicity of the drug needed for its incorporation in the oil particles even after intravenous injection was found to be clog P > 8, where clog P is the calculated logarithm of the partition coefficient between n-octanol and water. In the case of Sudan II (clog P = 5.4), the release from the oil particles was very quick after intravenous injection, resulting in slight alteration in biodistribution when compared with its micellar solution. In contrast, menatetrenone (clog P = 9.5) was selectively delivered to the liver, lungs and spleen, being consistent with the oil particles taken up by RES.

O/W Lipid Emulsions for Parenteral Drug Delivery. II. Effect of Composition on Pharmacokinetics of Incorporated Drug

The potential usefulness of O/W lipid emulsions as injectable drug delivery systems for lipophilic drugs was examined using a model lipophilic drug, sudan II (clogP = 5.4) in the normal rats. The standard lipid emulsion composed of soybean oil and egg yolk phosphatides increased the blood concentration of sudan II after i.v. injection when compared with its solubilized solution by plasma. However, it was still lower than that of the oil particles, and the distribution of sudan II to liver, lungs, adipose tissue, heart, and muscle was not altered, and only that to brain and kidneys was decreased. Herein, the effect of extensive alterations in the lipid emulsion composition on the blood concentration and organ distribution of sudan II was examined in comparison with the standard formulation. Addition of cholesterol, use of pure egg yolk phosphatidylcholine, use of phospholipids with saturated alkyl chain, use of saturated long chain triglycerides, and use of saturated medium chain triglycerides were tested. The oil particles of all tested lipid emulsions were still located in plasma space, and use of saturated medium chain triglycerides was the most effective way to increase blood concentration of sudan II, resulting in higher distribution to liver, lungs, spleen, and brain. This was caused by the increase of the steady-state partition of sudan II to the oil particles, and not by alteration of their organ distribution clearance.

Fat emulsions with added free cholesterol or fatty acid cholesteryl esters. Studies on removal mechanisms in vivo and hydrolysis by lipoprotein lipase in vitro.

The fractional elimination rate of fat emulsions of soybean oil, emulsified with egg yolk phosphatides with 1% addition of cholesterol or various cholesteryl fatty acid esters, was studied in rabbits. The fractional removal rate k2%/min was the same after addition of free cholesterol or esters with fatty acids up to eight carbon esters. The k2 values were twice as high for emulsions with cholesteryl-stearate, three times higher with added cholesteryl-palmitate and four times higher when cholesteryl-linoleate was added. The triglyceride (TG) lipase activity was determined with human or rabbit post-heparin plasma and with purified bovine lipoprotein lipase. All these enzyme sources gave similar results. Addition of saturated cholesteryl esters did not affect the lipase activity, but addition of 1% cholesterol markedly decreased the lipase activity. Furthermore, addition of cholesteryl-linoleate and linolenate reduced post-heparin TG lipase activity.

The influence of emulsifying agents and of lipid soluble drugs on the fractional removal rate of lipid emulsions from the blood stream of the rabbit.

Abstract: The fractional removal rate of intravenously injected fat emulsions prepared with various emulsifiers, was studied in rabbits. The emulsions were made with soya bean oil and addition of egg yolk phosphatides, acetylated monoglycerides or polyoxypropylene‐polyoxyethylene condensates (Pluronic). In some studies lipid soluble drugs were also added to the emulsions. The fractional removal rates of the emulsions from the blood stream were determined by means of an intravenous fat tolerance test. Addition of phosphatides slightly promoted the elimination of the fat emulsions, whereas the surface active agent Pluronic F 108 considerably impaired the fractional removal rate. Addition of cyclandelate or nitroglycerin increased the fractional elimination rate, whereas diazepam and chlorprozamine had the opposite effect. The half‐life of secobarbital in plasma was unaffected whether administered as emulsion or water solution, but the half‐life of thiopental was slightly longer when given as an emulsion. The pharmacological effect of barbiturates measured as duration of sleep in mice was not affected whether these agents were dissolved in slowly or rapidly eliminated emulsions. The results support the concept that the emulsifying agent is of major importance for the fractional removal rate of the emulsion from the blood stream, and further that the pharmacological effect of barbiturates dissolved in emulsions of different types is not related to the removal rate of the emulsion particles.

L'Alimentation Parentérale.

One of the essential requisites of the treatment of catabolism inducing diseases is the maintenance of an adequate nutritional status. When this cannot be achieved by the usual means (normal or forcible feeding), it is necessary to have recourse to parenteral nutrition. An adequate supply of nitrogen, ions, water and calories can either totally replace or complete the feeding and restore a satisfactory nutritional equilibrium, even in cases of cachectic diseases. The nutritional program must comply with the following criteria: 1. The caloric needs of the patient, which can be considerable, have to be met: an average of 2,500 to 4,000 calories per day including 12 to 20 gm nitrogen. 2. At least 10% of this amount must be given as carbohydrates. 3. The nitrogen is supplied by protein hydrolysate or synthetic amino-acid perfusions. The optimal proportions between all the amino-acids (essentials and non essentials) must be accurately kept to. In order to achieve the best protein anabolism, 150 to 300 calories should be given per gm of nitrogen perfused. This may be achieved by associating carbohydrates with the protein perfusions and by simultaneous perfusion of a lipid emulsion or of a concentrated glucose preparation. 4. Two to three gm lipids per kilo per day should be administered. Some lipid emulsions are well tolerated (soya oil emulsified by egg yolk phosphatides) and make a considerable caloric contribution possible. 5. The needs of water and ions have to be adapted to each individual case. Vitamins are an essential part of prolonged parenteral nutrition. Parenteral nutrition is frequently indicated. It is practically inocuous. However, care is needed when administering lipid emulsions to hepatic patients. It is to be noted that the high caloric lipid emulsions or protein containing solutions at present available in Belgium are not those generally indicated. The introduction into our country of soya oil emulsions as well as new protein solutions better balanced in the relative proportions of the different amino-acids as well as in their nitrogen-caloric ratio will be a notable step forward in what has been hitherto a neglected aspect of the treatment: the maintenance of the patient in optimal nutritional status.

The ultrastrucutre of lipid particles in emulsions prepared with various emulsifiers.

SummaryLipid particles of soya bean oil emulsions prepared with various emulsifying agents (egg yolk phosphatides, acetylated monoglycerides and poly‐oxypropylene‐polyoxyethylene polymers) were examined in sections of osmium‐fixed pellets. The mean particle size fell within the range of 0·3 to 0·8 μm for all but one emulsion and did not appear to be related to the emulsifying system used.Morphologically, particles of emulsions containing phosphatides were coated by an electron dense granular surface layer which was absent with the other emulsifiers. The presence of drugs in the oil phase did not alter the appearance of the lipid particles.

Creaming of phosphatide stabilized fat emulsions by electrolyte solutions

Creaming effects of electrolytes on emulsions of cottonseed and soybean oils have been investigated. The emulsions were stabilized with egg yolk phosphatides or with soybean phosphatides of different degrees of purity. The differences in creaming of the emulsions by the electrolyte solutions apparently were due to the differences in composition or purity of the phosphatide emulsifiers, and to the differences in properties of the respective oils. Emulsions of cottonseed or soybean oils stabilized with chromatographically homogeneous lecithin creamed to a greater extent than did emulsions of the same oils stabilized with impure phosphatides. In the latter, emulsions of cottonseed oil were slightly less creamed than emulsions of soybean oil. Creaming effects of electrolytes on emulsions of cottonseed and soybean oils have been investigated. The emulsions were stabilized with egg yolk phosphatides or with soybean phosphatides of different degrees of purity. The differences in creaming of the emulsions by the electrolyte solutions apparently were due to the differences in composition or purity of the phosphatide emulsifiers, and to the differences in properties of the respective oils. Emulsions of cottonseed or soybean oils stabilized with chromatographically homogeneous lecithin creamed to a greater extent than did emulsions of the same oils stabilized with impure phosphatides. In the latter, emulsions of cottonseed oil were slightly less creamed than emulsions of soybean oil.

Design of an intravenous diet of amino-acids and fat suitable for intensive patient-care.

In serious illnesses complicated by dysphagia, vomiting, gastro intestinal obstruction, or ileus it is not possible to maintain nutrition by means of an adequate diet given by mouth or naso-gastric tube. The metabolic needs must therefore be supplied intravenously. Water and electrolytes are readily given, but it has been difficult t? satisfy the calorie and nitrogen requirements. Failure to provide these may not adversely influence the course of an illness of moderate severity or short duration, but during severe or prolonged illnesses, especially those associated with trauma or sepsis, the demands for calories and nitrogen are increased. Unless these demands are satisfied the patient will show muscle-wasting, delayed tissue repair, and a negative nitrogen balance ; the latter precedes the clinical signs. Nitrogen may be given intravenously as amino-acids, and commercial preparations have been available in this country since 1958. A high intake of calories in a relatively small volume can be provided conveniently only by a fat-emulsion (Moore, 1959). Until recently the emulsions available have produced serious toxic effects, but one prepared from soya bean oil and egg-yolk phosphatides (Intralipid) has been exten sively tested and has caused few serious reactions (Wretlind, 1964).