Artificial Chylomicrons Research Articles

Novel First-Generation Dissolution Models to Investigate the Release and Uptake of Oral Lymphotropic Drug Products.

Conventional dissolution tests only assess the aqueous release of drugs to ensure quality and performance, without indicating whether absorption occurs through the portal or the lymphatic circulation. To address this issue, this study aimed to develop novel first-generation dissolution models that could investigate the release and uptake of oral lymphotropic drugs and examine relevant formulation issues. Dissolution of three commercial lymphotropic drug products (Terbinafina, Apo-terbinafine, and Lamisil) was done using modified versions of USP Apparatus II and IV. The developed models contained a lymphatic compartment filled with artificial chylomicrons to account for absorption through intestinal lymphatic pathway. The various products exhibited different release profiles into the aqueous media and the lymphatic media across the two tested models. The modified USP IV apparatus demonstrated greater distinction in aqueous release patterns. However, the release pattern into the lymphatic media remained similar in both models. This work represents a progress in meeting the challenges posed by the increasing complexity of pharmaceutical products containing lipophilic drugs or formulations, and has the potential to contribute towards the development of in-vitro bioequivalence standards for formulations targeting intestinal lymphatics.

In Vitro Predictive Model for Intestinal Lymphatic Uptake: Exploration of Additional Enhancers and Inhibitors.

Drug absorption via chylomicrons holds significant implications for both pharmacokinetics and pharmacodynamics. However, a mechanistic understanding of predicting in vivo intestinal lymphatic uptake remains largely unexplored. This study aimed to delve into the intestinal lymphatic uptake of drugs, investigating both enhancement and inhibition using various excipients through our previously established in vitro model. It also examined the applicability of the model by assessing the lymphatic uptake enhancement of a lymphotropic formulation with linoleoyl polyoxyl-6 glycerides using the same model. The model successfully differentiated among olive, sesame, and peanut oils in terms of lymphatic uptake. However, it did not distinguish between oils containing long-chain fatty acids and coconut oil. Coconut oil, known for its abundance of medium-chain fatty acids, outperformed other oils. This heightened uptake was attributed to the superior emulsification of this oil in artificial chylomicron media due to its high content of medium-chain fatty acids. Additionally, the enhanced uptake of the tested formulation with linoleoyl polyoxyl-6 glycerides underscored the practical applicability of this model in formulation optimization. Moreover, data suggested that increasing the zeta potential of Intralipid® using sodium lauryl sulfate (SLS) and decreasing it using (+/-) chloroquine led to enhanced and reduced uptake in the in vitro model, respectively. These findings indicate the potential influence of the zeta potential on intestinal lymphatic uptake in this model, though further research is needed to explore the possible translation of this mechanism in vivo.

Development of a Novel In Vitro Model to Study Lymphatic Uptake of Drugs via Artificial Chylomicrons.

The lymphatic system plays a crucial role in the absorption of lipophilic drugs, making it an important route for drug delivery. In this study, an in vitro model using Intralipid® was developed to investigate the lymphatic uptake of drugs. The model was validated using cannabidiol, halofantrine, quercetin, and rifampicin. Remarkably, the uptake of these drugs closely mirrored what would transpire in vivo. Furthermore, adding peanut oil to the model system significantly increased the lymphatic uptake of rifampicin, consistent with meals containing fat stimulating lymphatic drug uptake. Conversely, the inclusion of pluronic L-81 was observed to inhibit the lymphatic uptake of rifampicin in the model. This in vitro model emerges as a valuable tool for investigating and predicting drug uptake via the lymphatic system. It marks the first phase in developing a physiologically based predictive tool that can be refined further to enhance the precision of drug interaction predictions with chylomicrons and their subsequent transport via the lymphatic system. Moreover, it can be employed to explore innovative drug formulations and excipients that either enhance or hinder lymphatic drug uptake. The insights gained from this study have significant implications for advancing drug delivery through the lymphatic system.

Chylopericardium or contamination by injectable lipid emulsion?

Effusions can show some surprises. We document the case of a fourteen-month-old male patient with short-bowel syndrome, hospitalized in a cardiology unit, receiving a chronic parenteral nutrition by a Broviac® catheter. The patient presented several thrombosis following iterative catheter replacements. In parallel with superior vena cava plasty, a right intra-atrial Broviac® catheter was placed in the absence of other peripheral venous accesses. This device has a cutaneous exit site to allow for infusion of a hyperosmolar lipid emulsion. Seven days later, a milky liquid was secreted from pericardial/mediastinal redon. A gel lipoprotein electrophoresis of the fluid suggested a preliminary diagnosis of chylopericardium. However, biochemical testing of certain analytes evoked a parenteral nutrition-related pericardial effusion and a possible pseudochyloperitoneum caused by the shearing of a migrated Broviac® in pericardium. The patient, on a fat-free diet, was admitted to the ICU to drain the effusion and reposition the catheter, with success. In the light of new datas on the interference of parenteral lipid emulsions with the lipoproteins gel electrophoresis, we will try to determine whether the apparent presence of chylomicrons in the gel would be the sign of a lesion of the lymphatic system, or rather the result of a contamination by artificial chylomicron in the lipid emulsion, if not the sign of contaminated blood. In our article, we highlight several considerations in identifying and confirming cases of pericardial effusion, such as chylopericardium and parenteral nutrition-related one, as well as points concerning the use of lipid emulsions for pediatric patients with short-bowel syndrome.

Quantification of Lipoprotein Uptake in Vivo Using Magnetic Particle Imaging and Spectroscopy.

Lipids are a major source of energy for most tissues, and lipid uptake and storage is therefore crucial for energy homeostasis. So far, quantification of lipid uptake in vivo has primarily relied on radioactive isotope labeling, exposing human subjects or experimental animals to ionizing radiation. Here, we describe the quantification of in vivo uptake of chylomicrons, the primary carriers of dietary lipids, in metabolically active tissues using magnetic particle imaging (MPI) and magnetic particle spectroscopy (MPS). We show that loading artificial chylomicrons (ACM) with iron oxide nanoparticles (IONPs) enables rapid and highly sensitive post hoc detection of lipid uptake in situ using MPS. Importantly, by utilizing highly magnetic Zn-doped iron oxide nanoparticles (ZnMNPs), we generated ACM with MPI tracer properties superseding the current gold-standard, Resovist, enabling quantification of lipid uptake from whole-animal scans. We focused on brown adipose tissue (BAT), which dissipates heat and can consume a large part of nutrient lipids, as a model for tightly regulated and inducible lipid uptake. High BAT activity in humans correlates with leanness and improved cardiometabolic health. However, the lack of nonradioactive imaging techniques is an important hurdle for the development of BAT-centered therapies for metabolic diseases such as obesity and type 2 diabetes. Comparison of MPI measurements with iron quantification by inductively coupled plasma mass spectrometry revealed that MPI rivals the performance of this highly sensitive technique. Our results represent radioactivity-free quantification of lipid uptake in metabolically active tissues such as BAT.

Vitamin A Absorption Determined in Rats Using a Plasma Isotope Ratio Method

ABSTRACTBackgroundBetter methods are needed for determining vitamin A absorption efficiency.ObjectiveOur objective was to measure vitamin A absorption in rats by adapting a plasma isotope ratio method previously used to determine cholesterol absorption.MethodsMale Sprague-Dawley rats [n = 14; 340 ± 16 g (mean ± SD)] received an oral tracer dose of [3H]retinyl acetate in oil plus an intravenous dose of [14C]vitamin A–labeled lymph prepared in a donor rat that had received [14C]retinyl acetate intraduodenally. Blood samples were collected on days 1, 2, 3, 6, 9, and 12, and plasma was analyzed for 3H and 14C; vitamin A absorption was calculated for each sample as (fraction of oral dose/fraction of intravenous dose) × 100. Radioactivity was also measured in feces and urine collected as pools on days 3, 6, 9, and 12 and in liver and remaining carcass on day 12.ResultsVitamin A absorption calculated as the plasma isotope ratio was >100% on day 1, 78% ± 5% on day 6, 76% ± 5% on day 9, and 74% ± 5% on day 12; fitting the data to an exponential function plus a constant predicted an absorption of 75% by day 14. Recovery of the oral dose in feces (day 0 to day 6) was low (6.2% ± 0.84%, n = 10) and the mean isotope ratio in day 9–12 urine pool was lower than that in plasma.ConclusionsThe plasma isotope ratio holds promise for estimating vitamin A absorption, but additional work is needed to determine how long studies need to be and if the doses should be administered simultaneously. For application of this method in humans, artificial chylomicrons labeled with a stable isotope of retinyl acetate could be used for the intravenous dose, with a different isotope required for the oral dose.

Chylomicron remnants: mediators of endothelial dysfunction?

Vascular disease is initiated by activation of the endothelium characterized by the predominance of pro-inflammatory and pro-coagulant changes in endothelial cells (ECs) referred to collectively as 'endothelial dysfunction'. There is increasing evidence that lipoproteins of dietary origin modulate EC function and the use of artificial chylomicron remnant-like particles (CRLPs) in vitro is now beginning to shed light on the molecular mechanisms through which these particles influence cell behaviour. CRLPs enriched in n-6 PUFAs (polyunsaturated fatty acids) influence the production of vasoactive mediators by ECs in a pro-inflammatory manner. Thus CRLPs reduce the synthesis and release of nitric oxide and alter the balance of release of vasodilator versus vasoconstrictor eicosanoids. These changes are accompanied by induction of cyclo-oxygenase-2 expression and activity as well as increased expression of adhesion molecules and the antioxidant defence enzyme haem oxygenase-1. CRLPs also activate a number of intracellular signalling pathways, including NF-kappaB (nuclear factor kappaB) and MAPKs (mitogen-activated protein kinases), which may be involved in mediating their effects on gene expression. The effects of CRLPs on EC behaviour can also be modulated by the type of fat/oxidation status of the particles. These findings support the hypothesis that lipoproteins of dietary origin directly regulate molecular events in the vascular wall.

Gadolinium incorporated reconstituted chylomicron emulsion for potential application in tumor neutron capture therapy

Gadolinium (Gd) neutron capture therapy (NCT) is currently under development as a potential approach for tumor therapy. Nanoparticles have been suggested as a potential delivery system to carry or target Gd to tumors for thermal or epithermal neutron irradiation. The reconstituted chylomicron emulsion is an artificial chylomicron remnant prepared using commercially available natural and biocompatible lipids. We proposed to use this nanometer-scale emulsion to deliver Gd to solid tumors by modifying the surface of the emulsion. A lipophilic Gd compound, gadolinium acetylacetonate (GdAcAc), was incorporated into the emulsion, resulting in a final pure Gd concentration of more than 1 mg/mL. The apparent solubility of GdAcAc was enhanced by about 6000-fold by this incorporation. The emulsion particles were shown to be stable in a two-week short-term stability study when stored at 4 °C. In addition, no extensive particle aggregation was observed when the emulsion particles were incubated in simulated biological media such as serum. Also, GdAcAc does not significantly ‘leak’ out from the emulsion particles. Only ∼5% was released in 20 h in a SDS (0.5% w/v) in phosphate buffered saline (pH 7.4, 10 mM) medium. Finally, the emulsion particles were coated with polyethylene glycol (PEG), and injected into Balb/C mice via the tail vein. A significant proportion (71.6±18.4%) of the PEG-coated, GdAcAc-incorporated emulsion remained circulating in the blood 5 h after the injection, while the PEG-free emulsion was mainly accumulated inside the liver. This chylomicron emulsion may be used to deliver Gd into solid tumors for NCT.

Targeting primaquine into liver using chylomicron emulsions for potential vivax malaria therapy

Primaquine (PQ) exerts a broad spectrum of activities against various stages of parasitic malaria. It remains as the only drug that destroys late hepatic stages and latent tissue forms of Plasmodium vivax and Plasmodium ovale. However, systems that can target PQ to liver hepatocytes, where malarial sporozoites reside, are needed to minimize the dose-limiting severe toxicities and side-effects caused by PQ. Recently, a reconstituted artificial chylomicron emulsion was generated using commercially available lipids and was shown to be preferentially taken up by liver hepatocytes following intravenous injection. We proposed to target PQ to hepatocytes by incorporating it into this chylomicron emulsion. We have shown that lipophilized PQ can be readily incorporated into the chylomicron emulsion. The PQ remained inside the emulsion without significant release. Moreover, PQ incorporated inside the emulsion was more stable than free PQ when incubated in serum. Finally, when intravenously injected into mice, the PQ-incorporated chylomicron emulsion led to significantly enhanced accumulation of PQ in liver, when compared to the injection of free PQ. This emulsion could be developed into a promising delivery system to target PQ into hepatocytes for vivax malaria therapy.

Intralipid 10%: physicochemical characterization

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010–1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be ≤3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Acute in vivo chylomicron metabolism and postalimentary lipoprotein alterations in normolipidemic male smokers

Increased postprandial lipemia has been stated as one of the mechanisms responsible for atherogenesis in smokers. We measured the postalimentary lipid response and the in vivo intravascular delipidation index of an artificial chylomicron emulsion in healthy adult smokers and controls. The blood was collected in the fasting state immediately after the smokers smoked one cigarette. The lipemia was measured 2, 4, 6 and 8 h postalimentarily in smokers (S, n=8) and in non-smoking controls (C, n=8) and the chylomicron metabolism rate was measured 2, 4, 6, 8, 12, 16, 20, 24 and 30 min after the injection of an artificial emulsion to S ( n=10) and to C ( n=10). The lipoproteins were isolated in the fasting period and 4 h after the fatty meal and their chemical composition in cholesterol, triglycerides, phospholipids and protein was determined. Smokers showed an increased lipolysis percentage value (mean±S.E.M.) of the artificial chylomicron (39.1±3.1) compared to controls (26.5±3.3) and higher levels of HDL 2-PL: 28.4±4.3 (S) versus 16.2±2.0 (C) mg/dl (mean±S.E.M.). In conclusion, the oral fat tolerance was not altered in smokers but an upregulation of the rate of metabolism of the TG-rich lipoproteins was elicited immediately after smoking one cigarette.

Metabolism of artificial chylomicrons emulsion on sedentary women after physical training program

Metabolism of artificial chylomicrons emulsion on sedentary women after physical training program

Structure and metabolic fate of triacylglycerol- and phospholipid-rich particles of commercial parenteral fat emulsions.

The lipid emulsions used in parenteral nutrition are constituted of particles rich in triacylglycerols (TAG) called artificial chylomicrons (200-500 nm in diameter; monolayer of phospholipids [PL] enveloping a TAG core) and PL-rich particles called liposomes (diameter inferior to 80 nm; bilayer of PL around an aqueous phase), which represent the excess emulsifier. Introduced into the circulation, the two populations of particles come into contact with circulating lipoproteins and cell membranes and experience the same overall fate: exchanges and transfers of lipids and apolipoproteins, enzymatic hydrolysis of TAG and PL, and internalization by different tissues. The relative importance of these different metabolic processes varies depending on the type of particle. The artificial chylomicrons undergo a hydrolysis of their TAG by lipoprotein lipase, with a release of fatty acids and formation of smaller particles of remnants, which are rapidly removed by the liver. In delivering fatty acids to the tissue, artificial chylomicrons fulfill an energy transport function similar to the natural chylomicrons. The liposomes hold little energy interest, and they also have deleterious effects when infused in excess. They inhibit the lipolysis of artificial chylomicrons and, by actively capturing endogenous cholesterol, they stimulate tissue cholesterogenesis and accumulate in the blood as lipoprotein-X, a long-lived abnormal lipoprotein. To limit as much as possible the metabolic perturbations due to the intravenous administration of exogenous PL, the emulsion has to be infused at a low rate, and should contain the minimal amount of excess PL.

4.P.227 Effect of dietary cholesterol on the metabolism of the artificial chylomicrons in young men

4.P.227 Effect of dietary cholesterol on the metabolism of the artificial chylomicrons in young men

Hepatic lipase may act as a ligand in the uptake of artificial chylomicron remnant-like particles by isolated rat hepatocytes.

Active and heat-inactivated hepatic lipase stimulated to a statistically comparable extent the uptake of chylomicron remnant-like particles by isolated rat hepatocytes by 3-fold and 2.3-fold respectively and, likewise, their binding to hepatic plasma membranes by 5-fold and 4-fold respectively. Hepatic lipase may facilitate uptake of these particles, not only as a lipolytic enzyme, but also as a ligand anchored to extracellular glycosaminoglycans.

Cholesteryl esters in lymph chylomicrons: contribution from high density lipoprotein transferred from plasma into intestinal lymph

Most of the cholesterol in intestinal chyle and chylomicrons is derived from plasma. Our aim was to determine how much plasma low density (LDL) and high density (HDL) lipoproteins contribute to the cholesterol in chyle and chylomicrons, and to examine how plasma cholesterol becomes associated with lymph chylomicrons. Intravenous injection of radioiodinated plasma lipoproteins into two chyluric patients showed that 82% of the HDL plasma pool transferred daily to intestinal chyle, corresponding to 58% of lymph cholesterol; LDL contributed 18% of its plasma pool, corresponding to 18% of lymph cholesterol. When plasma HDL radiolabeled in both the protein and cholesteryl ester moieties was injected, the isotope ratios of plasma HDL and lymph lipoproteins were identical; 85% of the HDL cholesteryl esters transferred to triglyceride-rich lipoproteins, while the apolipoproteins remained largely (70%) in the higher density lipoproteins of the chyle. Incubations of similarly labeled plasma HDL showed preferential transfer of cholesteryl esters to artificial chylomicrons mediated by a factor present in lipoprotein-free plasma. Thus, a sizable portion of plasma HDL enters intestinal lymphatics probably as intact HDL, and then transfers part of their cholesteryl esters to chylomicrons, possibly mediated by transfer proteins. Reverse cholesterol transport may therefore include an extravascular loop via lymph chylomicrons and chylomicron remnants to the liver.

Effect of dietary fish oil on the rate of very low density lipoprotein triacylglycerol formation and on the metabolism of chylomicrons.

The mechanism by which omega 3 fatty acids lower plasma triacylglycerol levels was investigated. Rats were fed fish oil, olive oil (10% fat by weight) or a nonpurified diet (4% fat by weight) for 15 days. Lipoprotein lipase was inhibited by intra-arterial administration of Triton WR 1339 to estimate hepatic triacylglycerol output. Rats fed the olive oil diet showed a higher rate of triacylglycerol formation than rats fed the omega 3 fatty acid diet or the low-fat diet. All three groups showed identical rates of removal from plasma of intraarterially administered artificial chylomicrons that had simultaneously been labeled with cholesteryl [1-14C]oleate and [9,10(n)-3H]triolein. Liver radioactivity and total fat content were lowest in rats fed the fish oil diet, indicating that omega 3 fatty acids were preferentially metabolized in liver. Chylomicrons obtained from donor rats fed either fish oil containing [14C]cholesterol or olive oil containing [3H]cholesterol were removed at similar rates when infused together intraarterially into recipient animals. A slower formation of plasma very low density lipoprotein triacylglycerols in rats fed fish oil is probably due to a faster rate of oxidation of the fatty acid chains in the liver resulting in decreased plasma triacylglycerol concentrations.

Oral estradiol-17β raises the level of plasma high-density lipoprotein in menopausal women by slowing down its clearance rate

Plasma lipoprotein composition, plasma kinetics of autologous [125I]HDL and the metabolism of iv administered radioactively labelled artificial chylomicrons were studied in postmenopausal women during a control period and after 4 months of oral estradiol-17 beta treatment (1 mg/m2 body surface per day). Drug treatment significantly raised plasma HDL-cholesterol levels (19%) in the fasting state and total apolipoprotein A-I (16%), but did not interfere with triglyceride, VLDL, LDL or apolipoprotein-B values. As compared with the control period, estradiol-17 beta administration significantly slowed down plasma [125I]HDL clearance by about 82% and reduced the delipidation index of the injected artificial chylomicrons by 47% as a consequence of impaired plasma lipolytic activity.

Independent regulation of chylomicron lipolysis and particle removal rates: Effects of insulin and thyroid hormones on the metabolism of artificial chylomicrons

The processes of chylomicron lipolysis and removal from plasma were investigated by the intra-arterial infusion of doubly labeled artificial chylomicrons in rats. The rate of lipolysis was measured as a delipidation index (DI), which is the glyceryl-tri 9,10( N)- 3H oleate ( 3H-TO) fraction removed from the particle as fatty acids, whereas the cholesteryl(1- 14C) oleate ( 14C-CO) plasma disappearance rate measures the splanchnic organ particle uptake. In the alloxan-diabetic rats, despite a normal DI, the 14C-CO plasma residence time (RT) was longer than in control animals and remained longer after stimulation of the lipoprotein lipase by heparin. DI and 14C-CO removal rate were not significantly altered by insulin administration to glucose-supplemented control rats. Lipolysis was remarkable in propylthiouracil (PTU)-induced hypothyroidism, and yet the 14C-CO removal rate was retarded. In hypothyroidism, heparin enhanced the 14C-CO removal more than in the control group; however, after heparin, the 14C-CO RT still remained higher in the hypotyroid animals as compared with the control group. Hyperthyroidism lowered the DI; nevertheless, the 14C-CO disappearance rate was faster than in controls. In summary, lack or excess of thyroid hormone influences both the chylomicron lipolysis and removal systems, whereas lack of insulin impairs mostly the particle removal from plasma, and excess of insulin has no effect on the chylomicron metabolism.

The use of liposomes for the preparation of protein-free lipid emulsions models of chylomicron remnants

Artificial chylomicron remnants were investigated as a new drug carrier system for the targeting of hepatic parenchymal cells. The emulsions presented here are similar in particle size and composition to natural lipoproteins. The preparations contained triolein, phospholipid, cholesterol and cholesteryl oleate. Egg yolk lecithin was either used to form multilamellar or unilamellar liposomes or it was incorporated into a lipid film prior to emulsification. Typically the lipid film contained triolein, cholesterol and cholesteryl oleate. When multilamellar liposomes were used however, cholesterol and cholesteryl oleate were incorporated into the vesicles. The emulsions were prepared by ultrasonication or by means of a microemulsifier. The unilamellar liposomes used with the microemulsifier yielded the best particle distribution, i.e. in the range of 40-60 as determined by quasi-elastic light scattering. The advantage of the method results from the complete emulsification of the components. The particle size remained unchanged during storage, although flocculation was observed. The results show that the synthesis of artificial chylomicron remnants in a microemulsifier is possible and reproducible.