LIPID EMULSIONS

A lipid emulsion is provided comprising water, an emulsifier, and a glyceride

oil component. The weight ratio of the emulsifier to glyceride oil is
approximately 0.04 to about 0.01. It has been found that intravenous lipid
emulsions having a weight ratio of emulsifier to glyceride oil of
approximately 0.04 to about 0.01 are more rapidly metabolically utilized.

Lipid emulsion is used as a model compound of a plasma lipoprotein particle

and it is applied to the drug delivery system. For example, chylomicron is a
complex in which soluble apolipoproteins are bound to lipid emulsion. We
found for the first time that emulsion could bind apolipoproteins (apoA-I,
apoCs, and apoE) about 10-fold more than liposome. In consequence, the
systemic catabolism of the particulates and their interaction with the cultured
cell change remarkably. In addition, we understand the mechanism by which
apolipoproteins recognize the topology of lipid surface, and we now are
studying its application to the delivery. Furthermore, we seek for the
mechanism by which cholesterol, cholesteryl ester and sphingomyelin serve
as risk factors of myocardial infarction and arteriosclerosis, the diseases
known to be related to the quality of plasma lipoproteins.

Schematic representation of lipid emulsions and liposomes.

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Preparation of Lipid emulsions.
A lipid mixture was prepared with 97% (w}w) triolein (95%pure; Sigma),
2±5% egg phosphatidylcholine (95% pure; Prolabo, Paris, France) and 0±5%
free cholesterol (99% pure; Sigma) at a ®nal concentration of 16% (w}w) in
chloroform} methanol (2 :1, v}v). Aliquots of the lipid mixture solution (1 ml
each) were evaporated to dryness under nitrogen in 7 ml glass tubes. The
buffer (with or without ®bre) was added to a ®nal volume of 4 ml. The
aqueous mixture thus contained 4% (w}w) lipids and 0±2% ®bre, i.e. in the
physiological range [5,6,23,24]. The tubes were stoppered, attached
horizontally and shaken at 200 strokes}min for 2 h at 37 °C. These operating
conditions resulted from preliminary experiments designed to discover
conditions that would generate lipid emulsions with droplet sizes in the range
found in human and rat stomach during fat digestion.

Emulsification measurements
Determination of the amount of emulsi®ed lipids The upper limit for
emulsion droplet size was set at 100 lm, given the instability and negligible
interfacial area of lipid droplets above this value [6]. To allow accurate
measurements, [carboxyl- "%C]triolein (98% pure; 69 mCi}mmol; CEA,
Gif-sur-Yvette, France) was added in trace amounts to the lipid mixture.
Radioactivity was measured by liquid scintillation counting using a Packard
1600TR instrument (Packard, Meriden, CT, U.S.A.) from 100 ll aliquots of
two fractions collected at the end of the emulsi®cation process. One fraction
was the ¯oating oily layer composed of unemulsi®ed lipids (oily material
plus lipid droplets &100 lm) which collect above the aqueous solution
surface when tubes are left to stand for a given time (0±5±10 min at 1 g)
calculated from the Stoke's sedimentation equation for droplets &100 lm
under the present conditions, as previously reported. According to Stoke's
sedimentation law, the relation between sedimentation time and particle
diameter is expressed in the following equation: where D is the particle
diameter (m), g! is the viscosity coefficient of the solvent (N[s}m#), q is the
density of the sample (kg}m$), q! is the density of the solvent (kg}m$), g is
acceleration due to gravity (m[s−#), t is the sedimentation time (s) and H is

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the distance of sedimentation (m). The second fraction was the resulting
infranatant containing emulsifed lipid droplets.

Measurement of droplet size

The distribution of the emulsion droplet sizes in the infranatant solution was
determined by using a particle-size analyser (Capa 700; Horiba, Kyoto,
Japan) as previously described [6,8,9,10,11]. The validity of the method has
previously been evaluated and calibrations made using microparticles in the
size range 0±2±100 lm (polystyrene size standard kit ; Polyscience Inc.,
Warrington, PA, U.S.A.). Measurements were carried out using gradient-
mode analysis at a constant centrifuge acceleration rate (960 rev.}min) to
allow an accurate measurement of large dropFibres and fat emulsions 271 lets
(100 lm) as well as small droplets (0±1 lm). The particle-sizer software
calculated the droplet-size distribution which is expressed as a fraction of the
total droplet volume. Results are given as a frequency-distribution graph
characterized by its median diameter (lm) and a speci®c interfacial area
(m#}g). The droplet surface area (m#) was calculated from the amount (g) of
emulsi®ed lipids in the infranatant solution.

Effects of fibres on lipid emulsification

To determine the effects of

soluble ®bres on the extent of
fat emulsi®cation, we measured
the amount of emulsi®ed lipid
and the size of the emulsi®ed
lipid droplets produced and
calculated the surface area
generated. Lipid emulsi®cation
occurred to a moderate extent
(24±6%) under the conditions
selected in the absence of ®bre
(control). Adding ®bres did not
markedly change this. For
instance, 0±3% solutions of the
®bres caused the following
extents of emulsi®cation : gum arabic, 23±8%; HVG, 29±7%; MVG,
25±6%; LVG, 24±2%; NND pectin, 27±8%; BNF pectin, 26±1%. The data

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obtained from droplet-size measurements of emulsi- ®ed lipids are given in
Table 2. In control buffer, the median diameter exhibited by the emulsion was
7±19 lm. Gum Arabic (0±3±2±0%), LVG and MVG at low concentrations
did not signi®cantly alter the droplet size. In contrast, LVG at 2±0%, MVG
at 0±6% and HVG at 0±3% signi®cantly increased the median size of the
droplets (45±6, 41±7 and 25±4 lm respectively). Also, BNF pectin and NND
pectin signi®cantly increased the droplet median diameter to a comparable
extent at concentrations of 0±6% and 0±8% respectively. A comparison of the
effects of the ®bres tested at a given concentration (0±3%; Table 2) provides
the following information: the highest median diameters were elicited by
HVG (25±41³4±09 lm) and to a lesser degree MVG (14±36³4±78 lm) and
NND pectin (15±42³4±34 lm) whereas the other ®bres [LVG (8±71³0±29 lm)

Emulsion Test For Lipids

Lipids do not dissolve in water, but do dissolve in ethanol. This characteristic
is used in the emulsion test.

⇒ Grind up sample

⇒ Shake some test sample with about 4cm3 of ethanol.

⇒Decant the liquid into a test tube of water leaving any un-dissolved
substances between.

If there are lipids dissolved in the ethanol, they will precipitate in the water,
forming a cloudy white emulsion.

Lipid Emulsions: Formation, Stability, and Metabolism

Lipid emulsions composed of triacylglycerol (TG) and phosphatidylcholine
(PC) have been commonly used for parenteral nutrition for almost 30 years.
Recently, lipid emulsions were used as drug carriers in drug-delivery
systems.1 For example, the delivery of prostaglandin E1 in injectable lipid
emulsions has been well established and is commercially available.2, 3 Lipid
emulsions are classified as macroemulsions and thermodynamically unstable,
which may bring about aggregation, flocculation, coalescence, and eventual

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phase separation over time. It is generally believed that freezing or freeze-
drying lipid emulsion formulations is an effective form of storage after
production. However, there have been problems with coalescence of
emulsion droplets during the freeze-thawing (F-T) procedures. When
emulsions freeze, the lipid droplets become progressively concentrated and
come into contact with one another in unfrozen aqueous channels between
the ice crystals. The condensation of the lipid droplets in the narrow channels
could lead to aggregation as well as coalescence in the F-T process.4, 5
Emulsion types, O/W or W/O, and their stability have been predicted by the
Bancroft rule: The phase in which the emulsifier has higher solubility tends
to be the continuous outer phase. Numerous exceptions to this rule have been
noted. For example, PC has much larger solubility in TG than aqueous
medium; however, these mixtures are emulsified to give O/W lipid
emulsions. PC also stabilizes water droplets in methyloleate.6

Lipid emulsions containing droplets that are 100 nm in diameter and single-
surface monolayers of zero net charge, i.e., with similar hydrodynamic and
electrical properties, were prepared. First, the stability of the lipid droplets
against the coalescence in the highly concentrated state [in the floating cream
(ultracentrifugation) and in the unfrozen aqueous channels between the ice
crystals (F-T processes)] was evaluated with changing surface and core lipid
components. The results were discussed on the basis of the coalescence
transition state theory recently developed by Kabalnov and collegues.7, 8 The
theory predicts that an emulsifier with bulky alkyl chains and a small polar
head group (surface lipid with negative spontaneous curvature) stabilizes
W/O emulsions well, whereas an emulsifier with small alkyl chains and a
bulky polar head group (surface lipid with positive spontaneous curvature)
effectively stabilizes O/W emulsions.7, 8

In animals, lipid emulsions are rapidly hydrolyzed by lipoprotein lipase

(LPL) and the remnants are readily taken up by the liver after intravenous
injection. Because the release of drugs in blood is considered to depend on
both the lipolysis of TG and the clearance of the particles from plasma, it is
important to control these metabolic processes in order to improve lipid
emulsions as injectable drug carriers. Emulsion droplets in this sudy have a
size and lipid composition similar to chylomicrons and have also been used
as models for plasma lipoproteins. The metabolism of protein-free lipid
emulsions in rats is comparable to that of chylomicrons.9, 10 On entering the
plasma, lipid emulsions rapidly acquire apolipoproteins, such as apoA-I, C-II,
and E, from circulating lipoproteins. It is thought that apoC-II is an activator
of LPL, and apoE is necessary for recognition by lipoprotein receptors. On
the other hand, an inhibitory effect on hepatic uptake through apoE-specific
receptors by apoCs (C-I, C-II, and C-III) has been reported.11-1611, 12, 13,

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14, 15, 16 The metabolism of lipid emulsions can be affected by the selective
binding of these apolipoproteins to lipid particles. Within a blood
compartment, TG-rich lipoproteins are converted into remnants through the
hydrolysis of TG by LPL.17 These remnants are subsequently taken up by
the liver, although hepatic removal appears to be accomplished by several
overlapping mechanisms.18 Lipolysis of TG in chylomicrons produces Chol-
enriched remnant particles.19 It is presumed that the amount of Chol in the
lipoprotein surface is an important factor influencing the metabolism of
lipoproteins. In addition, differences in sphingomyelin (SM) content among
plasma lipoproteins may affect both lipolysis by LPL at endothelial sites and
recognition by lipoprotein receptors.20-2420, 21, 22, 23, 24 However, little
has been elucidated on the physiological role of SM in lipoprotein
metabolism. We assume that the lipid composition of lipoproteins or lipid
emulsions plays a crucial role in the metabolism of the particles. In other
words, it must be possible to regulate both clearance from plasma and triolein
lipolysis of artificial lipid emulsions by modulating the lipid composition of
an emulsion surface. In this article we evaluate the effects of Chol and SM in
the emulsion surface on the lipolysis and clearance from plasma in rats. The
selectivity of apolipoprotein binding was estimated and its relevance to
plasma clearance is discussed. These results are useful both for the deeper
understanding of lipoprotein metabolism and for the development of
improved lipid-emulsion drug carriers.

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 APPLICATIONS OF LIPID EMULSIONS

Use of lipid emulsion in the resuscitation of a patient

with prolonged cardiovascular collapse after overdose of
bupropion and lamotrigine.

Animal studies show efficacy of intravenous lipid emulsion in the treatment

of severe cardiotoxicity associated with local anesthetics, clomipramine, and
verapamil, possibly by trapping such lipophilic drugs in an expanded plasma
lipid compartment ("lipid sink"). Recent case reports describe lipid infusion
for the successful treatment of refractory cardiac arrest caused by parenteral
administration of local anesthetics, but clinical evidence has been lacking for
lipid's antidotal efficacy on toxicity caused by ingested medications. A 17-
year-old girl developed seizure activity and cardiovascular collapse after
intentional ingestion of up to 7.95 g of bupropion and 4 g of lamotrigine.
Standard cardiopulmonary resuscitation for 70 minutes was unsuccessful in
restoring sustained circulation. A 100-mL intravenous bolus of 20% lipid
emulsion was then administered, and after 1 minute an effective sustained
pulse was observed. The patient subsequently manifested significant acute
lung injury but had rapid improvement in cardiovascular status and
recovered, with near-normal neurologic function. Serum bupropion levels
before and after lipid infusion paralleled triglyceride levels. This patient
developed cardiovascular collapse because of intentional, oral overdose of
bupropion and lamotrigine that was initially refractory to standard
resuscitation measures. An infusion of lipid emulsion was followed rapidly
by restoration of effective circulation. Toxicologic studies are consistent with
the lipid sink theory of antidotal efficacy.

Lipid Emulsions in Parenteral Nutrition

Lipid emulsions containing a physical mixture of medium and long chain
triglycerides (MCT/LCT) are a well-proven concept in parenteral nutrition of
critically ill patients. Having a demonstrably higher utilization rate,
MCT/LCT emulsions do not impair liver function, produce less immune and
no reticuloendothelial system function compromise, and do not interfere with

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pulmonary hemodynamics or gas exchange. A reduced content of n-6
polyunsaturated fatty acids can also be obtained by using newer preparations
based on structured triglycerides or olive oil. Further studies are necessary in
order to investigate these new lipid emulsions versus the physical mixture of
MCT/LCT. A promising substrate in the development of lipid emulsions can
be seen in fish oils. With regard to current literature, fish oils have a
beneficial influence on the pathophysiological response to endotoxins and
exert important modulations on eicosanoid and cytokine biology.
Furthermore their intravenous use may improve organ perfusion in different
critical situations.

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 REFRENCE:

WEBSITES:

1. www.pharmainfo.net

2. http://content.karger.com/

3. http://www.ncbi.nlm.nih.gov

4. http://www.freepatentsonline.com/

5. http://www.pubmedcentral.nih.gov/

6. http://www.informaworld.com/