U.S. patent application number 13/038832 was filed with the patent office on 2011-06-23 for structured triglycerides and emulsions comprising same.
Invention is credited to Geila Rozen, Irit Shochat.
Application Number | 20110150944 13/038832 |
Document ID | / |
Family ID | 34919503 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110150944 |
Kind Code |
A1 |
Rozen; Geila ; et
al. |
June 23, 2011 |
STRUCTURED TRIGLYCERIDES AND EMULSIONS COMPRISING SAME
Abstract
The present invention relates to structured triglycerides, to
parenteral nutrition emulsions of the same, and uses thereof. In
particular, the invention relates to structured triglycerides that
include at least one medium chain C.sub.6-C.sub.12 fatty acid and
at least one fatty acid selected from long chain C.sub.14-C.sub.18
or very long chain C.sub.20-C.sub.22 fatty acids. Preferably, each
fatty acid is present in a predetermined position of the glycerol
backbone. The parenteral nutrition emulsions are particularly
useful for nourishing preterm- and term-infants, children,
critically ill patients, and cancer patients.
Inventors: |
Rozen; Geila; (Haifa,
IL) ; Shochat; Irit; (Timrat, IL) |
Family ID: |
34919503 |
Appl. No.: |
13/038832 |
Filed: |
March 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10591734 |
May 1, 2007 |
7919526 |
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PCT/IL2005/000257 |
Mar 3, 2005 |
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13038832 |
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60549550 |
Mar 4, 2004 |
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Current U.S.
Class: |
424/400 ;
435/134; 514/458; 514/552; 554/124; 554/227 |
Current CPC
Class: |
C12P 7/6472 20130101;
A61P 3/02 20180101; A61P 31/18 20180101; C11C 3/08 20130101; A23L
33/115 20160801; A23L 33/12 20160801; A23D 7/0053 20130101; C11C
3/10 20130101; A61P 37/02 20180101; A61K 31/23 20130101; A61P 43/00
20180101; A23D 7/011 20130101; A23D 7/003 20130101 |
Class at
Publication: |
424/400 ;
554/227; 514/552; 514/458; 554/124; 435/134 |
International
Class: |
A61K 31/355 20060101
A61K031/355; C07C 57/02 20060101 C07C057/02; A61K 31/232 20060101
A61K031/232; A61K 9/107 20060101 A61K009/107; C07C 67/00 20060101
C07C067/00; A61P 31/18 20060101 A61P031/18; A61P 37/02 20060101
A61P037/02; A61P 43/00 20060101 A61P043/00; C12P 7/64 20060101
C12P007/64 |
Claims
1. A structured triglyceride comprising a glycerol backbone having
three fatty acid residues esterified thereto, wherein two fatty
acid residues selected from the group consisting of
C.sub.6-C.sub.12 fatty acids and active derivatives thereof are
esterified to the internal position and to a first external
position of the triglyceride backbone, and one fatty acid residue
selected from the group consisting of C.sub.14-C.sub.18 fatty
acids, C.sub.20-C.sub.22 fatty acids, and active derivatives
thereof is esterified to a second external position of the
triglyceride backbone.
2. The structured triglyceride according to claim 1, wherein the
C.sub.14-C.sub.18 fatty acids are selected from the group
consisting of myristic acid, palmitic acid, palmitoleic acid,
stearic acid, oleic acid, linoleic acid, alpha linolenic acid, and
any combination thereof.
3. The structured triglyceride according to claim 1, wherein the
C.sub.20-C.sub.22 fatty acids are selected from the group
consisting of arachidonic acid, eicosapentaenoic acid,
docosahexaenoic acid, and any combination thereof.
4. The structured triglyceride according to claim 1, wherein the
C.sub.14-C.sub.18 fatty acids and the C.sub.20-C.sub.22 fatty acids
are selected from the group consisting of .omega.-3, .omega.-6,
.omega.-9 fatty acids, and any combination thereof.
5. A parenteral nutrition emulsion composition comprising a
structured triglyceride according to claim 1.
6. The parenteral nutrition emulsion composition according to claim
5, wherein the structured triglyceride is present in an amount of
about 80% to about 100% of total triglycerides of said
emulsion.
7. The parenteral nutrition emulsion composition according to claim
5, wherein the C.sub.14-C.sub.18 fatty acids and the
C.sub.20-C.sub.22 fatty acids are selected from the group
consisting of .omega.-3, .omega.-6, .omega.-9 fatty acids, and any
combination thereof.
8. The parenteral nutrition emulsion composition according to claim
5, comprising from about 9% to about 90% by weight C.sub.6-C.sub.12
fatty acids based on the weight of total fatty acids.
9. The parenteral nutrition emulsion composition according to claim
5, comprising from about 9% to about 90% by weight
C.sub.14-C.sub.18 fatty acids based on the weight of total fatty
acids.
10. The parenteral nutrition emulsion composition according to
claim 5, comprising from about 1% to about 10% by weight
C.sub.20-C.sub.22 fatty acids based on the weight of total fatty
acids.
11. The parenteral nutrition emulsion composition according to
claim 7, wherein the .omega.-6 fatty acids and the .omega.-3 fatty
acids are in a ratio of about 7:1 to about 1:1.
12. The parenteral nutrition emulsion composition according to
claim 5, wherein the structured triglyceride constitutes from about
10% to about 40% (w/v) of the composition.
13. The parenteral nutrition emulsion composition according to
claim 5, wherein a droplet size of said emulsion is lower than
about 1 .mu.m.
14. The parenteral nutrition emulsion composition according to
claim 5, further comprising tocopherol.
15. The parenteral nutrition emulsion according to claim 14,
wherein the tocopherol is alpha tocopherol.
16. The parenteral nutrition emulsion according to claim 5, further
comprising at least one component selected from the group
consisting of emulsifiers, surfactants, carbohydrates, vitamins,
amino acids, trace minerals, osmolality modifiers and water.
17. The parenteral nutrition emulsion according to claim 5
comprising: (a) 20% (w/v) structured triglycerides comprising:
about 40-50% by weight C.sub.6-C.sub.12 fatty acids based on the
weight of total fatty acids, wherein the C.sub.6-C.sub.12 fatty
acids comprise 0-5% caproic acid, 20-30% caprylic acid, 10-30%
capric acid, and 0-5% lauric acid by weight based on the weight of
total fatty acids; about 35-55% by weight C.sub.14-C.sub.18 fatty
acids based on the weight of total fatty acids, wherein the
C.sub.14-C.sub.18 fatty acids comprise 0-5% mystiric acid, 5-30%
palmitic acid, 0-5% palmitoleic acid, 0-5% stearic acid, 10-30%
oleic acid, 10-30% linoleic acid, and 5-15% alpha linolenic acid by
weight based on the weight of total fatty acids; and about 1-10%
C.sub.20-C.sub.22 by weight fatty acids based on the weight of
total fatty acids, wherein the C.sub.20-C.sub.22 fatty acids
comprise 1-5% AA, 0-5% EPA, and 1-5% DHA by weight based on the
weight of total fatty acids, wherein the ratio of .omega.-6 to
.omega.-3 fatty acids is 1:1-2:1; (b) 1.2% (w/v) phospholipids; (c)
1.8-2.0 mg/1 g of fatty acids alpha tocopherol; (d) 0-25 g/L
glycerin; and (e) water.
18. The parenteral nutrition emulsion composition according to
claim 5 comprising: (a) about 20% (w/v) structured triglycerides
comprising: about 45% by weight C.sub.6-C.sub.12 fatty acids based
on the weight of total fatty acids, wherein the C.sub.6-C.sub.12
fatty acids comprise 2.5% caproic acid, 30% caprylic acid, 10%
capric acid, and 2.5% lauric acid by weight based on the weight of
total fatty acids; about 50% by weight C.sub.14-C.sub.18 fatty
acids based on the weight of total fatty acids, wherein the
C.sub.14-C.sub.18 fatty acids comprise 10% palmitic acid, 2.5%
stearic acid, 15% oleic acid, 16% linoleic acid, and 7% alpha
linolenic acid by weight based on the weight of total fatty acids;
and about 5% by weight C.sub.20-C.sub.22 fatty acids based on the
weight of total fatty acids, wherein the C.sub.20-C.sub.22 fatty
acids comprise 1.5% AA, 1.5% EPA, and 1.5% DHA by weight based on
the weight of total fatty acids, wherein the ratio of .omega.-6 to
.omega.-3 fatty acids is 1.75; (b) about 1.2% (w/v) phospholipids;
(c) about 1.8 mg/1 g of fatty acids alpha tocopherol; (d) about
10-25 g/L glycerin; and (e) water.
19. A process of synthesizing a structured triglyceride according
to claim 1 comprising the step of performing an acidolysis
reaction.
20. The process according to claim 19, wherein the acidolysis
reaction is catalyzed by a lipase.
21. The process according to claim 19, wherein the triglyceride is
a medium chain triglyceride.
22. The process according to claim 19, wherein the fatty acid is
selected from the group consisting of C.sub.14-C.sub.18 fatty
acids, C.sub.20-C.sub.22 fatty acids, and active derivatives
thereof.
23. The process according to claim 19 further comprising a step of
distilling the reaction mixture to remove non-reacted medium chain
triglyceride and fatty acid.
24. A process of preparing a parenteral nutrition emulsion
composition according to claim 5 comprising the step of reducing
the droplet size of the emulsion below of about 1 .mu.m.
25. A method of providing nutrition to a subject in need thereof
comprising parenterally administering to the subject a parenteral
nutrition emulsion composition according to claim 5.
26. The method according to claim 25, wherein the subject is a
preterm infant, a term infant, a child, an adult, a critically ill
patient, a cancer patient, or a patient suffering from one of the
conditions selected from trauma, burns, malnutrition, starvation,
aging, and immunosuppression.
27. The method according to claim 25, wherein the subject is an
AIDS patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/591,734, filed May 1, 2007, which is the 371 national state of
International application PCT/IL2005/000257 filed Mar. 3, 2005,
which claims the benefit of application No. 60/549,550 Mar. 4,
2004. The entire content of each earlier filed application is
expressly incorporated herein by reference thereto.
FIELD OF INVENTION
[0002] The present invention relates to structured triglycerides,
to emulsions comprising same suitable for parenteral nutrition, and
use thereof. In particular, the invention relates to structured
triglycerides comprising at least one medium chain C.sub.6-C.sub.12
fatty acid and at least one fatty acid selected from long chain
C.sub.14-C.sub.18 and very long chain C.sub.20-C.sub.22 fatty
acids, preferably each fatty acid being in a predetermined position
of the glycerol backbone. The parenteral nutrition emulsions are
particularly useful for nourishing preterm and term infants,
children, critically ill patients, and cancer patients.
BACKGROUND OF THE INVENTION
[0003] Lipids have been used as an integral component of parenteral
nutrition over the last four decades. Lipids provide essential
fatty acids for cellular structures, specifically cell membranes,
and for precursors of prostaglandins, leukotrienes, thromboxanes
and other eicosanoids. They constitute a source of energy, take
part in various biosynthetic pathways, and are carriers of
fat-soluble vitamins. As such, lipids play an important role in
metabolic and immune processes, in the development and function of
the central nervous system and the retina.
[0004] Fatty acids (FA) differ from one another by the number of
carbon atoms, their saturation or degree of non-saturation, the
positions of unsaturated bonds, and whether these bonds are cis or
trans. All of these variables are relevant to the nutritional value
or benefit derived from triglycerides containing these acids. In
addition, the enzymatic cleavage of the triglycerides is affected
by the type and position of the fatty acids on the glycerol
backbone.
[0005] Fatty acids in general are divided into four groups: short
chain FA, medium chain FA (MCFA), long chain FA (LCFA), and very
long chain FA (VLCFA). Fatty acids are also classified by the
presence, number, and location of double bonds. This classification
divides FA into three groups: saturated FA (no double bonds),
monounsaturated FA (one double bond) and polyunsaturated FA (2
double bonds and more). Further classification of the
polyunsaturated FA is characterized by the placement of the carbon
preceding the first double bond from the terminal methyl carbon:
n-3 or .omega.-3 FA, n-6 or .omega.-6 FA and n-9 or .omega.-9 FA.
These differences determine the various characteristics of FA and
therefore their specific functions.
Lipid Emulsions
[0006] The need for lipids as essential and integral component of
parenteral nutrition (PN) emerged from the observations of the
clinical symptoms following use of fat free PN. These clinical
symptoms included hemorrhagic dermatitis, skin atrophy,
hyperglycemia, weight loss, decrease of immune function, increase
of catabolism, etc.
[0007] The first generation of lipid emulsions was based on pure
long chain triglycerides (LCT) derived from soybean oil and
safflower oil. Their administration prevented some of the symptoms
of fatty acid deficiency. Nevertheless, patients that received
these lipid emulsions showed impaired function of lymphocytes and
of the reticuloendothelial system, depressed T-cell counts,
increased oxygen free radical production, elevation of liver
enzymes, hypertriglyceridemia, and suffered from infections.
[0008] The next generation of lipid emulsions contained 50% medium
chain triglycerides (MCT) and 50% LCT. These emulsions have many
advantages compared to pure LCT emulsions, for example, they are an
efficient energy source, they are more soluble, rapidly hydrolyzed
by lipases, quickly eliminated from blood, rapidly oxidized, and
have smaller particle size. As the MCFA are all saturated, they are
not subjected to peroxide formation and therefore they do not
impair the immune and reticuloendothelial systems. Patients
receiving MCT/LCT emulsions demonstrate a better nitrogen balance
and a better protein sparing effect.
[0009] Another attempt to overcome the disadvantages of pure LCT
emulsions was to use olive oil, rich in monounsaturated oleic acid
(18:1 .omega.-9). Olive oil based emulsions were shown to be
well-tolerated, more suitable for preventing lipid peroxidation,
and maintained a normal essential FA status. It was also
demonstrated that olive oil emulsions contain primarily alpha
tocopherol, the more biologically active tocopherol, while soybean
oil emulsions contain predominantly gamma tocopherol, which has
little protection against lipid peroxidation. When the composition
and peroxidation of lipoproteins were compared in children
receiving olive oil or soybean oil emulsions, it was found that
administration of olive oil emulsions resulted in a decreased
oxidative stress. Gobel et al., and Goulet et al., showed the
advantages of olive oil emulsions compared to other LCT emulsions
in preterm infants and in children (Gobel Y., et al., J. Pediatr.
Gastroenterol. Nutr. 37(2): 161-167, 2003; Goulet, O., et al., Am.
J. Clin. Nutr. 70(3): 338-345, 1999). Oleic acid, and in general
the .omega.-9 fatty acids, have been shown to contribute to brain
development and function as they are a major component of the white
matter and myelin.
[0010] The beneficial effects of .omega.-3 fatty acids derived from
fish oil in enteral feeding prompted their inclusion in parenteral
nutrition. The most successful regimen was achieved by the
combination of 50% MCT, 40% soybean oil and 10% fish oil. This
regimen demonstrated an improvement in the immune system function
of surgical and critically ill patients, an improvement of FA
profile in cell membranes, anti-inflammatory and anti-coagulation
effects, a normalization of plasma triglycerides (TG) and
cholesterol, and a reduction in blood pressure.
Structured Triglycerides
[0011] Lipid emulsions containing randomized structured
triglycerides (STG) have been obtained by mixing MCT and LCT oils
and heating the mixture in the presence of a catalyst. During this
process, MCFA and LCFA can be exchanged randomly on the glycerol
backbone of both oils. The new TG thus formed contains both long
and medium chain FA on the same glycerol, randomly distributed.
This kind of triglycerides are rapidly hydrolyzed by lipases, and
hence are better cleared from the blood stream.
[0012] Many clinical studies have demonstrated the safety and the
advantages of STG emulsions. Sandstrom et al., demonstrated that
STG emulsions administered to postoperative patients were rapidly
cleared from the plasma, rapidly oxidized, and were not associated
with any side effects (Sandstrom, R., et al., JPEN 19 (5): 381-386,
1995). Provision of STG caused a significantly higher whole body
fat oxidation compared to LCT. Rubin et al, demonstrated that STG
appear to be safe and well tolerated on a long term basis in
patients on home parenteral nutrition and suggested that STG
emulsions may be associated with possible reduction in liver
dysfunction (Rubin, M., et al., Nutrition, 16: 95-100, 2000).
[0013] Kruimel et al., compared the effect of STG versus physical
mixture of MCT and LCT on the nitrogen balance of moderately
catabolic postoperative patients. Over a period of 5 days the
cumulative nitrogen balance was less negative in the STG group
(Krumiel, J. W., et al., JPEN 25(5): 237-244, 2001). This
difference can be explained by better utilization of the STG fatty
acids for energy and better clearance from the blood (ibid.).
Chambrier et al., compared the effect of STG vs. a physical mixture
of MCT-LCT on liver function in postoperative patients. A
significant increase in liver enzymes and in plasma TG was found to
occur in patients administered with the physical mixture of
MCT-LCT, while no changes in liver function nor in plasma TG level
were found to occur in patients administered with STG (Chambrier,
C., et al., Nutrition, 15: 274-277, 1999).
[0014] U.S. Pat. No. 4,871,768 discloses a synthetic triglyceride
comprising a glycerol backbone having three fatty acids attached
thereto, wherein at least one fatty acid is selected from .omega.3
fatty acids and at least one fatty acid is selected from
C.sub.8-C.sub.10 fatty acids. The .omega.3 fatty acids are derived
from plant oils, marine plankton oils, fungal oils, or fish oils.
U.S. Pat. No. 4,871,768 also discloses a dietary supplement
comprising 10 to 40% by weight of an oily fraction, the oily
fraction comprises 10 to 90% by weight of the synthetic
triglyceride. The synthetic triglyceride in the dietary supplement
according to U.S. Pat. No. 4,871,768 further comprises .omega.9
fatty acids. Yet, the necessity of docosahexaenoic acid (DHA) and
arachidonic acid in the synthetic triglyceride, and the necessity
of vitamin E in the dietary supplement have not been indicated nor
the optimal ratio of .omega.6 to .omega.3.
[0015] U.S. Pat. No. 4,906,664 discloses a method for providing
nutritional support to patients suffering from cancer cachexia. The
method comprises the step of parenteral administration of a diet
containing a structured lipid. The structured lipid according to
U.S. Pat. No. 4,906,664 is a triglyceride wherein at least one of
the chains is a medium chain fatty acid, at least one of the chains
is an .omega.3 long chain fatty acid, and the other chain is
selected from the group consisting of medium chain fatty acids and
long chain fatty acids. The ratio of long chain fatty acids to
medium chain fatty acids is about 1:1. The long chain fatty acids
should be primarily .omega.3 and .omega.6 fatty acids, with
sufficient .omega.6, preferably in the form of linoleic acid.
[0016] U.S. Pat. No. 5,081,105 discloses a method of treating
sarcomas in a patient through the use of nutritional support
therapy comprising the step of parenterally administering a diet
including a structured lipid. The structured lipid according to
U.S. Pat. No. 5,081,105 is a triglyceride where one of the chains
is a medium chain fatty acid, a second chain is a .omega.3 fatty
acid, and the third chain is selected from H, OH, short, medium,
and long fatty acids.
[0017] U.S. Pat. No. 5,962,712 discloses a family of structured
lipids, one of the fatty acid residues is selected from the group
consisting of gamma linolenic acid (GLA) and dihomogamma linolenic
acid (DHGLA). A second fatty acid residue is selected from
C.sub.18-C.sub.22 n-3 fatty acids, and the third fatty acid residue
is selected from C.sub.6-C.sub.12 fatty acids. The simultaneous
presence of C.sub.18-C.sub.22 n-3 fatty acid residues and GLA or
DHGLA may serve to minimize the elongation of GLA and DHGLA to
arachidonic acid. The long chain polyunsaturated n-3 fatty acids
will purportedly shift the prostaglandin metabolism away from
pro-inflammatory prostanoids to non-inflammatory prostanoids,
having beneficial effects in treating inflammation and infection.
U.S. Pat. No. 5,661,180 discloses a method of modulating metabolic
response to trauma and disease states in a patient comprising the
step of administering a dietary structured lipid as disclosed in
U.S. Pat. No. 5,962,712.
[0018] There is an unmet need for structured triglycerides designed
to provide improved enteral or parenteral nutrition, which is
easily assimilated by infants, children, and patients suffering
severe stress or chronic illness and which is optimized to address
developmental and immunological needs.
SUMMARY OF THE INVENTION
[0019] It is now disclosed that parenteral nutrition emulsions
comprising structured triglycerides comprising medium chain (MCFA),
long chain (LCFA), and very long chain fatty acid residues (VLCFA),
are highly advantageous for parenteral nutrition, particularly for
preterm- and term-infants, children, critically ill patients, and
cancer patients. The present invention provides parenteral
nutrition emulsions comprising structured triglycerides having
specific beneficial ratios of MCFA, LCFA and VLCFA.
[0020] The present invention further provides parenteral nutrition
emulsions comprising structured triglycerides wherein the position
of the fatty acid residues on the glycerol backbone is
predetermined.
[0021] The present invention discloses for the first time
parenteral nutrition emulsions comprising structured triglycerides
comprising at least one MCFA, and at least one LCFA or VLCFA, the
LCFA or VLCFA is esterified primarily at the external position of
the glycerol backbone. According to some embodiments, the VLCFA are
selected from arachidonic acid (AA; 20:4 .omega.-6),
eicosapentaenoic acid (EPA; 20:5 .omega.-3), docosahexaenoic acid
(DHA; 22:6 .omega.-3), or any combination thereof. The parenteral
nutrition emulsions provide high nutritional advantage, improve the
immune system function, and have beneficial effects on the
structure and function of cell membranes, on the development and
function of the brain, CNS and retina, on the regulation of blood
pressure, and on coagulation processes. The parenteral nutrition
emulsions of the invention are, therefore, particularly beneficial
for preterm- and term-infants, children, cancer patients, and
critically ill patients.
[0022] As the structured triglycerides of the present invention
comprise at least one MCFA and at least one LCFA or VLCFA on the
same glycerol backbone, the physical characteristics of these
structured triglyceride emulsions are improved compared to those of
pure LCT or mixed LCT/MCT emulsions. Thus, the structured
triglyceride emulsions of the invention achieve lower particle size
than mixed LCT/MCT emulsions and consequently they can be filtered
through a filter of a pore size of 0.22 .mu.m. The structured
triglyceride emulsions of the invention are also more soluble and
more stable than mixed LCT/MCT emulsions. Additionally, as the LCFA
and the VLCFA are located preferably at the external position of
the glycerol backbone, the clearance of these fatty acids from the
blood is faster than if they were positioned on the internal
position, and therefore these structured triglycerides enable
maintenance of a regulated blood triglyceride level.
[0023] It is also disclosed that a low ratio of .omega.-6 to
.omega.-3 fatty acids, particularly an .omega.-6/.omega.-3 ratio
lower than 2:1 in the parenteral nutrition emulsions comprising the
structured triglycerides provides beneficial effects on brain
development in preterm- and term-infants, and in children. The low
.omega.-6/.omega.-3 ratio provides beneficial effects on the immune
system and on the heart function and these effects are particularly
essential in critically ill patients and in cancer patients. A
decrease of the .omega.-6 fatty acid intake and an increase of the
.omega.-3 fatty acid intake with no supplementation of sufficient
amounts of AA and DHA may impair various biological processes such
as blood coagulation cascades and regulation of blood pressure. It
may also impair the chemical and physical characters of cell
membranes, and the development and function of the brain, CNS, and
retina. Therefore, the presence of AA and DHA in the structured
triglycerides of the invention is of high importance both for
infants, children, and adult patients.
[0024] It is further disclosed that inclusion of monounsaturated
oleic acid (18:1 .omega.-9) in the structured triglycerides
provides superior properties compared to polyunsaturated fatty
acids, as the former is required for the structure and function of
the brain. In addition, oleic acid is less susceptible to peroxide
formation compared to polyunsaturated fatty acids, and therefore
inclusion of this fatty acid provides less exposure to peroxidation
damages.
[0025] It is also disclosed that addition of vitamin E,
particularly alpha tocopherol, to the parenteral nutrition
emulsions provides protection of the subject nourished with said
parenteral nutrition emulsions against peroxide formation, and
therefore protects the subject from peroxidation damages.
[0026] According to one aspect, the present invention provides a
structured triglyceride comprising a glycerol backbone having three
fatty acid residues esterified thereto, wherein at least one fatty
acid residue is selected from the group consisting of
C.sub.6-C.sub.12 fatty acids and active derivatives thereof, and at
least one fatty acid residue is selected from the group consisting
of C.sub.14-C.sub.18 fatty acids, C.sub.20-C.sub.22 fatty acids,
and active derivatives thereof, with the proviso that a
C.sub.18-C.sub.22 .omega.-3 fatty acid residue is not present on
the same glycerol backbone together with gamma linolenic acid or
dihomogamma linolenic acid.
[0027] According to another aspect, the present invention provides
a structured triglyceride comprising a glycerol backbone having
three fatty acid residues esterified thereto, wherein at least one
fatty acid residue is selected from the group consisting of
C.sub.6-C.sub.12 fatty acids and active derivatives thereof in the
internal position of the glycerol backbone, and at least one fatty
acid residue is selected from the group consisting of
C.sub.14-C.sub.18 fatty acids, C.sub.20-C.sub.22 fatty acids, and
active derivatives thereof in an external position of the glycerol
backbone.
[0028] According to some embodiments, the C.sub.14-C.sub.18 fatty
acids are selected from the group consisting of saturated,
monounsaturated, polyunsaturated fatty acids, and any combination
thereof. According to additional embodiments, the C.sub.14-C.sub.18
fatty acids are selected from the group consisting of myristic acid
(14:0), palmitic acid (16:0), palmitoleic acid (16:1), stearic acid
(18:0), oleic acid (18:1 .omega.-9), linoleic acid (18:2
.omega.-6), alpha linolenic acid (18:3 .omega.-3), and any
combination thereof.
[0029] According to other embodiments, the C.sub.20-C.sub.22 fatty
acids are selected from the group consisting of arachidonic acid
(AA; 20:4 .omega.-6), eicosapentaenoic acid (EPA; 20:5 .omega.-3),
docosahexaenoic acid (DHA; 22:6 .omega.-3), and any combination
thereof.
[0030] According to another aspect, the present invention provides
a parenteral nutrition emulsion composition comprising a structured
triglyceride, the structured triglyceride comprises a glycerol
backbone having three fatty acid residues esterified thereto,
wherein at least one fatty acid residue is selected from the group
consisting of C.sub.6-C.sub.12 fatty acids and active derivatives
thereof, and at least one fatty acid residue is selected from the
group consisting of C.sub.14-C.sub.18 fatty acids,
C.sub.20-C.sub.22 fatty acids, and active derivatives thereof, with
the proviso that a C.sub.18-C.sub.22 .omega.-3 fatty acid residue
is not present on the same glycerol backbone together with gamma
linolenic acid or dihomogamma linolenic acid.
[0031] According to a further aspect, the present invention
provides a parenteral nutrition emulsion composition comprising a
structured triglyceride, the structured triglyceride comprises a
glycerol backbone having three fatty acid residues esterified
thereto, wherein at least one fatty acid residue is selected from
the group consisting of C.sub.6-C.sub.12 fatty acids and active
derivatives thereof in the internal position of the glycerol
backbone, and at least one fatty acid residue is selected from the
group consisting of C.sub.14-C.sub.18 fatty acids,
C.sub.20-C.sub.22 fatty acids, and active derivatives thereof in an
external position of the glycerol backbone.
[0032] According to some embodiments, the parenteral nutrition
emulsion composition comprises from about 9 to about 90%
C.sub.6-C.sub.12 fatty acids based on the weight of total fatty
acids in the parenteral nutrition emulsion composition. According
to additional embodiments, the parenteral nutrition emulsion
composition comprises from about 30 to about 60% C.sub.6-C.sub.12
fatty acids based on the weight of total fatty acids in the
parenteral nutrition emulsion composition. According to further
embodiments, the parenteral nutrition emulsion composition
comprises from about 40 to about 50% C.sub.6-C.sub.12 fatty acids
based on the weight of total fatty acids in the parenteral
nutrition emulsion composition. According to non-limiting exemplary
embodiments, the parenteral nutrition emulsion composition
comprises caproic acid (6:0), caprylic acid (8:0), capric acid
(10:0), and lauric acid (12:0), which constitute, respectively,
about 0-5%, about 20-30%, about 10-30%, and about 0-5% by weight of
total fatty acids in the parenteral nutrition emulsion
composition.
[0033] According to other embodiments, the parenteral nutrition
emulsion composition comprises from about 9 to about 90%
C.sub.14-C.sub.18 fatty acids based on the weight of total fatty
acids in the parenteral nutrition emulsion composition. According
to additional embodiments, the parenteral nutrition emulsion
composition comprises from about 30 to about 70% C.sub.14-C.sub.18
fatty acids based on the weight of total fatty acids in the
parenteral nutrition emulsion composition. According to additional
embodiments, the parenteral nutrition emulsion composition
comprises from about 35 to about 55% C.sub.14-C.sub.18 fatty acids
based on the weight of total fatty acids. According to non-limiting
exemplary embodiments, the parenteral nutrition emulsion
composition comprises myristic acid, palmitic acid, palmitoleic
acid, stearic acid, oleic acid, linoleic acid, and alpha linolenic
acid, which constitute, respectively, about 0-5%, about 5-30%,
about 0-5%, about 0-5%, about 10-30%, about 10-30% and about 5-15%
by weight of total fatty acids in the parenteral nutrition emulsion
composition.
[0034] According to additional embodiments, the parenteral
nutrition emulsion composition comprises from about 1 to 20%
C.sub.20-C.sub.22 fatty acids based on the weight of total fatty
acids in the parenteral nutrition emulsion composition. According
to some embodiments, the parenteral nutrition emulsion composition
comprises from about 1 to about 10% C.sub.20-C.sub.22 fatty acids
based on the weight of total fatty acids in the parenteral
nutrition emulsion composition. According to non-limiting exemplary
embodiments, the parenteral nutrition emulsion composition
comprises AA, EPA, and DHA, which constitute, respectively, about
1-5%, about 0-5% and about 1-5% by weight based on the weight of
total fatty acids in the parenteral nutrition emulsion composition.
It will be understood that a combination of the fatty acids
disclosed hereinabove in a parenteral nutrition emulsion is highly
advantageous in order to provide the nutritional needs of preterm-
and term-infants, children, adults, cancer patients, patients
suffering from burns, and critically ill patients.
[0035] According to some embodiments, a majority of structured
triglycerides of the parenteral nutrition emulsion compositions of
the present invention have one fatty acid residue selected from the
group consisting of C.sub.14-C.sub.18 fatty acids,
C.sub.20-C.sub.22 fatty acids, and active derivatives thereof, and
two fatty acid residues selected from the group consisting of
C.sub.6-C.sub.12 fatty acids and active derivatives thereof.
According to additional embodiments, the structured triglycerides
having one fatty acid residue selected from the group consisting of
C.sub.14-C.sub.18 fatty acids, C.sub.20-C.sub.22 fatty acids, and
active derivatives thereof, and two fatty acid residues selected
from the group consisting of C.sub.6-C.sub.12 fatty acids and
active derivatives thereof comprise from about 80% to about 100% of
total structured triglycerides of said emulsions.
[0036] According to other embodiments, the ratio of
.omega.-6/.omega.-3 fatty acids in the parenteral nutrition
emulsion composition ranges from about 7:1 to 1:1. According to
additional embodiments, the ratio ranges from about 3:1 to about
1:1. According to an exemplary embodiment, the ratio of
.omega.-6/.omega.-3 fatty acids in the parenteral nutrition
emulsion composition ranges from about 2:1 to about 1.5:1. The
present invention thus also encompasses ratio of .omega.-6 to
.omega.-3 fatty acids of 1:1.
[0037] According to some embodiments, the parenteral nutrition
emulsion compositions comprise from about 10 to about 40% (w/v) of
the structured triglycerides of the invention. According to other
embodiments, the parenteral nutrition emulsion compositions
comprise from about 15 to about 30% (w/v) of the structured
triglycerides of the invention. According to an exemplary
embodiment, the parenteral nutrition emulsion compositions comprise
about 20% (w/v) of the structured triglycerides of the
invention.
[0038] According to additional embodiments, a droplet size of the
parenteral nutrition emulsion compositions of the invention is
lower than about 1 .mu.m. According to some embodiments, the
droplet size of the parenteral nutrition emulsion compositions of
the invention is lower than about 0.45 .mu.m. According to other
embodiments, the droplet size is lower than about 0.22 .mu.m. It
will be understood that this droplet size enables filtering the
emulsion through a membrane filter having a pore size of 0.22
.mu.m, and thus achieves higher sterilization of the emulsions.
[0039] According to other embodiments, the parenteral nutrition
emulsion composition further comprises vitamin E. According to some
embodiments, the vitamin E is alpha tocopherol. The amount of the
alpha tocopherol in a parenteral nutrition emulsion of the
invention is from about 0.1 to about 5 mg per 1 g of fatty acids.
Preferably, the amount of alpha tocopherol is from about 1 to about
2 mg per 1 g of fatty acids. According to an exemplary embodiment,
the parenteral nutrition emulsion comprises 1.8-2.0 mg of alpha
tocopherol per 1 g of fatty acids.
[0040] According to some embodiments, the parenteral nutrition
emulsion composition further comprises an emulsifier. The amount of
an emulsifier such as, for example phospholipids, in the parenteral
nutrition emulsion composition is from about 0.5 to about 4% (w/v).
According to additional embodiments, the amount of the emulsifier
is from about 0.5 to about 2.5% (w/v). According to an exemplary
embodiment, the parenteral nutrition emulsion composition comprises
about 1-1.2% (w/v) of phospholipids.
[0041] According to other embodiments, the parenteral nutrition
emulsion composition can further comprise an osmolality modifier.
An example of an osmolality modifier is glycerin. The amount of an
osmolality modifier can range from about 1 to about 5% (w/v).
[0042] The parenteral nutrition emulsion composition can further
comprise at least one component selected from the group consisting
of surfactants, carbohydrate nutrients, electrolytes, amino acids,
vitamins, trace minerals, preservatives and water. The parenteral
nutrition emulsion composition can also comprise sterile water.
[0043] According to certain non-limiting embodiments, the
parenteral nutrition emulsion composition comprises: [0044] (a)
about 20% (w/v) structured triglycerides comprising: [0045] about
40-50% by weight C.sub.6-C.sub.12 fatty acids based on the weight
of total fatty acids, wherein the C.sub.6-C.sub.12 fatty acids
comprise 0-5% caproic acid, 20-30% caprylic acid, 10-30% capric
acid, and 0-5% lauric acid based on the weight of total fatty
acids; [0046] about 35-55% by weight C.sub.14-C.sub.18 fatty acids
based on the weight of total fatty acids, wherein the
C.sub.14-C.sub.18 fatty acids comprise 0-5% mystiric acid, 5-30%
palmitic acid, 0-5% palmitoleic acid, 0-5% stearic acid, 10-30%
oleic acid, 10-30% linoleic acid, and 5-15% alpha linolenic acid
based on the weight of total fatty acids; and [0047] about 4.5-5.5%
by weight C.sub.20-C.sub.22 fatty acids based on the weight of
total fatty acids, wherein the C.sub.20-C.sub.22 fatty acids
comprise 1-5% AA, 0-5% EPA, and 1-5% DHA based on the weight of
total fatty acids, wherein the ratio of .omega.-6 to .omega.-3
fatty acids is about 1:1-2:1; [0048] (b) about 1.2% (w/v)
phospholipids; [0049] (c) about 1.8-2.0 mg/1 g of fatty acids alpha
tocopherol; [0050] (d) about 10-25 g/L glycerin; and [0051] (e)
water.
[0052] According to another exemplary embodiment, the parenteral
nutrition emulsion composition comprises: [0053] (a) about 20%
(w/v) structured triglycerides comprising: [0054] about 45% by
weight C.sub.6-C.sub.12 fatty acids based on the weight of total
fatty acids, wherein the C.sub.6-C.sub.12 fatty acids comprise 2.5%
caproic acid, 30% caprylic acid, 10% capric acid, and 2.5% lauric
acid by weight based on the weight of total fatty acids; [0055]
about 50% by weight C.sub.14-C.sub.18 fatty acids based on the
weight of total fatty acids, wherein the C.sub.14-C.sub.18 fatty
acids comprise 10% palmitic acid, 2.5% stearic acid, 15% oleic
acid, 16% linoleic acid, and 7% alpha linolenic acid by weight
based on the weight of total fatty acids; and [0056] about 5% by
weight C.sub.20-C.sub.22 fatty acids based on the weight of total
fatty acids, wherein the C.sub.20-C.sub.22 fatty acids comprise
1.5% AA, 1.5% EPA, and 1.5% DHA by weight based on the weight of
total fatty acids, wherein the ratio of .omega.-6 to .omega.-3
fatty acids is 1.75; [0057] (b) about 1.2% (w/v) phospholipids;
[0058] (c) about 1.8 mg/1 g of fatty acids alpha tocopherol; [0059]
(d) about 10-25 g/L glycerin; and [0060] (e) water.
[0061] According to a further aspect, the present invention
provides a process of synthesizing a structured triglyceride of the
invention comprising the step of performing an acidolysis reaction.
According to some embodiments, the acidolysis reaction is catalyzed
by a lipase. According to additional embodiments, the triglyceride
is a medium chain triglyceride. According to additional
embodiments, the fatty acid is selected from the group consisting
of C.sub.14-C.sub.18 fatty acids, C.sub.20-C.sub.22 fatty acids,
and active derivatives thereof. According to additional
embodiments, the process of synthesizing the structured
triglyceride of the invention further comprises the step of
distilling the reaction mixture to remove non-reacted MCT and fatty
acid.
[0062] According to another aspect, the present invention provides
a process of preparing a parenteral nutrition emulsion composition
comprising the step of reducing the droplet size of the emulsion
below of about 1 .mu.m. According to some embodiments, the droplet
size is reduced below 0.45 .mu.m. According to non-limiting
exemplary embodiments, the droplet size is reduced below 0.22
.mu.m.
[0063] According to another aspect, the present invention provides
a method of providing nutrition to a subject in need thereof
comprising parenterally administering to the subject a parenteral
nutrition emulsion composition of the invention.
[0064] According to some embodiments, the subject to be nourished
by the parenteral nutrition emulsion composition of the invention
is a preterm infant, a term infant, a child, an adult, a critically
ill patient, a cancer patient, or a patient suffering from surgical
trauma, burns, malnutrition, starvation, aging, or
immunosuppression. According to other embodiments, the subject to
be nourished by the parenteral nutrition emulsion of the invention
is a patient suffering from AIDS.
[0065] These and other embodiments of the present invention will be
better understood in relation to the description, examples, and
claims that follow.
BRIEF DESCRIPTION OF THE FIGURES
[0066] FIG. 1 shows a schematic presentation of the
lipase-catalyzed acidolysis reaction between MCT and free fatty
acids in solvent-free system. M represents medium-chain fatty acyl
group and Lc represents long-chain or very long chain fatty acyl
group.
[0067] FIGS. 2A-B show HPLC chromatograms of MCT before and after
an acidolysis reaction. FIG. 2A, peak 1 represents the free fatty
acid, namely palmitic acid while peaks 2, 3, 4 and 5 represent the
MCT before the reaction and peak 6 is trilaurin, an internal
standard. FIG. 2B, peaks 1, 2 and 3 represent the liberated
medium-chain fatty acids, while peak 4 represents the non-reacted
palmitic acid. Peaks 5, 6, 7 and 8 represent the non-reacted MCT in
the reaction medium, while peaks 9, 10, and 11 represent the new
products containing one long-chain fatty acyl group. Peaks 12 and
13 represent the new products containing two long-chain fatty acyl
groups.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention provides structured triglycerides and
parenteral nutrition emulsions comprising same useful in parenteral
nutrition of preterm- and term-infants, children, adults,
critically ill patients, and cancer patients.
[0069] According to one aspect, the present invention provides a
structured triglyceride comprising a glycerol backbone having three
fatty acid residues esterified thereto, wherein at least one fatty
acid residue is selected from medium chain fatty acids and active
derivatives thereof, and at least one fatty acid residue is
selected from the group consisting of long chain fatty acids, very
long chain fatty acids, and active derivatives thereof, with the
proviso that a C.sub.18-22 .omega.-3 fatty acid residue is not
present on the same glycerol backbone together with gamma linolenic
acid or dihomogamma linolenic acid.
[0070] The term "active derivatives" as used herein includes
esters, ethers, amines, amides, substituted fatty acids (e.g.,
halogen substituted fatty acids), and other substitutions, which do
not affect the beneficial properties of the fatty acids.
[0071] The term ".omega.-3", ".omega.-6" and ".omega.-9" as used
herein refers to a fatty acid in which a double bond is present at
the third carbon, sixth carbon, and ninth carbon, respectively,
from the methyl end of the hydrocarbon chain. This nomenclature is
equivalent to the n-3, n-6, and n-9 designations. Thus, the terms
.omega.-3, .omega.3, and n-3; .omega.-6, .omega.6, and n-6; and
.omega.-9, .omega.9, and n-9 are used interchangeably throughout
the specification and claims of the present invention.
[0072] The structured triglycerides of the invention are made as
"designer oils". Using enzymatic procedures known in the art that
direct the incorporation of specific fatty acids to specific
positions in the glycerol molecule, structured triglycerides are
synthesized.
[0073] U.S. Pat. No. 6,537,787, the content of which is
incorporated by reference as if fully set forth herein, discloses a
method for obtaining a mixture enriched with polyunsaturated fatty
acid triglycerides in the presence of a position-specific lipase,
particularly 1,3-specific lipase. The specific lipase according to
U.S. Pat. No. 6,537,787 is preferably a Candida antarctica
lipase.
[0074] U.S. Pat. No. 6,518,049, the content of which is
incorporated by reference as if fully set forth herein, discloses a
process for esterifying a marine oil composition containing EPA and
DHA as free fatty acids to form a free fatty acid fraction enriched
in at least one of these fatty acids as compared to the starting
composition, comprising the step of reacting the marine oil
composition with glycerol in the presence of a lipase catalyst
under reduced pressure and essentially organic solvent-free
conditions, and recovering a free fatty acid fraction enriched in
at least one of EPA and DHA. According to U.S. Pat. No. 6,518,049
the lipase is preferably immobilized on a carrier and is Rhizomucor
miehei lipase.
[0075] Sugihara et al. (Appl. Microbiol. and Biotech. 1993
40:279-83 and references cited therein) disclosed a microorganism
having a moderate selectivity towards the sn-2 position in
glycerides. Ota et al. (Biosci. Biotechnol. Biochem. 2000, 64:
2497) disclosed an enzyme of Geotrichum candidum, which hydrolyzes
the sn-2-positioned ester bond nearly twice more as compared to
hydrolysis of the 1- or 3-positioned ester bonds.
[0076] U.S. Pat. No. 6,605,452 to Basheer, the content of which is
incorporated by reference as if fully set forth herein, discloses a
lipase preparation immobilized onto an insoluble matrix that
preferably has 1,3-positional specificity with respect to
triacylglycerols.
[0077] Thus, according to the principles of the present invention,
a lipase can be used in a crude form, e.g., as supplied by a
manufacturer or as isolated by any method known in the art for
isolation of lipases, or a lipase or a surfactant-coated lipase
complex can be immobilized on an insoluble matrix and subsequently
used for preparing the structured triglycerides of the present
invention. The lipase can be derived from any source, though a
preferable source is a microorganism. Many different species can be
used as a source of the lipase including, but not limited to,
Aspergillus niger, Aspergillus oryzae, Burkholderia sp., Candida
antarctica such as Candida antarctica A and B, Candida cylindracea,
Candida lipolytica, Candida rugosa, Chromobacterium viscosum,
Humicola sp., Lipoprotein lipase Pseudomonas A, Mucor javanicus,
Mucor miehei, Penicillium roqueforti, Pseudomonas fluorescens,
Pseudomonas Cepacia, Porcine pancreatic lipase (PPL), Rhizopus
arrhizus, Rhizopus javanicus, Rhizopus japonicus, Rhizopus oryzae,
Rhizopus miehei, and Rhizopus niveus, Thermomyces lanuginose, and
Wheat germ lipase. The lipases used for the production of the
structured triglycerides of the invention have 1,3-positional
specificity with respect to the structured triglyceride.
[0078] Structured triglycerides of this invention can be prepared
by various methods as are well known in the art. The structured
triglycerides can be prepared by esterification of fatty acids and
glycerol, acidolysis, transesterification and interesterification.
In the specification and the claims that follow the term acidolysis
includes reactions between one or more free fatty acid and a
triglyceride to exchange at least one fatty acid of the
triglyceride with at least one of the free fatty acids. According
to some embodiments acidolysis is performed by reacting MCT and
free fatty acids (i.e., reaction of the free fatty acids with MCT
to exchange one or more of the MCFA). In the specification and the
claims that follow the term transesterification includes reactions
between two distinct triglycerides to exchange at least one fatty
acid of the first triglyceride with at least one of the fatty acids
of the second triglyceride. According to some embodiments
transeterification is performed by reacting an MCT and an LCT. In
the specification and the claims that follow the term
interesterification includes reactions of a triglyceride with an
alkyl ester of a fatty acid. According to some embodiments the
interesterification reaction of the fatty acid alkyl ester is with
an MCT to exchange one or more of the MCFA. The fatty acid alkyl
ester is typically though not exclusively a methyl or ethyl fatty
acid ester. According to some embodiments the reaction is catalyzed
using a lipase preparation. It should also be noted that contacting
a lipase preparation with fatty acid-containing substrates may be
effected within a reaction reactor, e.g., a tank reactor or a
fixed-bed reactor. It will be understood that any method known in
the art for preparing the structured triglycerides is encompassed
in the present invention.
[0079] Typically, the structured triglycerides of the invention can
be prepared by acidolysis reaction between MCT and free fatty acids
using lipases as supplied by the manufacturers or immobilized on an
insoluble matrix. For immobilization of the lipases, an insoluble
matrix, such as a silicate or an ion-exchange resin, is used and
the lipases are immobilized from their aqueous solutions. Wet
immobilized lipases are pretreated with a mixture of biphase medium
containing phosphate buffer solution and oil and then dried under
vacuum to reduce the water content of the immobilized lipase down
to less than 2 wt %. Lipases can also be immobilized and activated
according to procedures well known in the art. For example,
immobilized and activated lipases possessing acidolysis activity
can be prepared by attaching a polyethylene group to the surface
functional group of the enzyme according to U.S. Pat. No. 4,645,741
to Inada et. al. Lipases can also be activated and immobilized
according to the procedure described by Boosley et al. (U.S. Pat.
No. 5,232,843) where a lipase is immobilized on a hydrophobic
surface, e.g., silica or an ion-exchange resin, precoated with a
non-lipase protein. Immobilized lipases on a dry, porous
particulate hydrophobic support and containing a surfactant
prepared according to U.S. Pat. No. 5,773,266 and U.S. Pat. No.
6,596,520, can also be applied for producing the structured
triglycerides of the present invention.
[0080] Additionally or alternatively, other methods for preparing
structured triglycerides of the present invention can involve the
use of alkali metal catalysts such as sodium, and/or nucleophilic
catalysts, such as hydroxides and alkoxides, which function to
randomly exchange fatty acids on the glycerol backbone of the
glycerol molecule.
[0081] Inclusion of C.sub.6-C.sub.12 fatty acids in the structured
triglycerides has some benefits. The C.sub.6-C.sub.12 fatty acids
do not need carnitine to enter the mitochondria, thus they are
rapidly cleared from blood and are used as energy source. As a
component of the structured triglycerides, MCFA contribute to
achieve a lower molecular weight, better solubility and better
stability of the emulsion.
[0082] According to another aspect, the present invention provides
a parenteral nutrition emulsion comprising the structured
triglyceride of the invention. It is apparent to a person skilled
in the art that the present invention encompasses nutrition
emulsions comprising the structured triglycerides of the invention
for enteral nutrition.
[0083] According to some embodiments, the parenteral nutrition
emulsion further comprises vitamin E, preferably alpha tocopherol.
The normal range of plasma tocopherol concentrations is between 0.7
and 1.6 mg/100 ml. Generally, the recommended amount of vitamin E
for premature infants is 4.55 mg/day and for adults it is 100-200
mg/day. Vitamin E is classified as a practically non-toxic
substance. A dosage below 1000 mg/day is safe and free from side
effects. In order to maintain normal range of tocopherol, vitamin E
should be matched quantitatively to unsaturated FA. Therefore, the
parenteral nutrition emulsion of the invention can comprise 0.1 to
5 mg of alpha tocopherol per 1 g of fatty acids. Preferably, the
parenteral nutrition emulsion can comprise 1 to 2 mg of alpha
tocopherol per 1 g of fatty acids.
[0084] The parenteral nutrition emulsion composition according to
the invention can advantageously further comprise a natural
biologically compatible emulsifier. The emulsifier is preferably a
phospholipid compound or a mixture of phospholipids, such as
lecithin, phosphatidylcholine, phosphatidyl ethanolamine or
mixtures thereof. Non-limiting examples of phospholipids which can
be used in the compositions of the invention are lecithins;
EPIKURON 170.RTM. being a mixture of about 70% (w/v) of
phosphatidylcholine, 12% phosphatidylethanolamine, and about 16%
other phospholipids, or OVOTHIN 160.RTM. being a mixture comprising
about 60% (w/v) phosphatidylcholine, 18% (w/v)
phosphatidylethanolamine, and 12% (w/v) other phospholipids, both
manufactured by Lucas Meyer (Germany). These mixtures of mainly
phosphatidylcholine and phosphatidylethanolamine are derived from a
natural source, such as purified egg yolk phospholipids (for the
Ovothin series) and soybean oil phospholipids (for the Epikuron
series); a purified phospholipid mixture; LIPOID E-80.RTM. being a
phospholipid mixture comprising about 80% (w/v)
phosphatidylcholine, about 8% (w/v) phosphatidylethanolamine, about
3.6% non-polar lipids, and about 2% sphingomyeline, manufactured by
Lipoid KG (Ludwigshafen, FRG). Other phospholipids of plants (e.g.,
lecithin) or of animal origin known in the art can be used as
emulsifiers for the preparations of the parenteral nutrition
emulsion compositions of the invention. For example, other forms of
emulsifiers containing fatty acyl groups, such as polyol fatty acid
esters, can be used for the preparations of such emulsions.
[0085] The emulsion can further comprise a pharmaceutically
acceptable non-natural surfactant. Any conventional
pharmaceutically acceptable non-ionic surfactant can be used.
Generally, the surfactant is a non-ionic alkylene oxide condensate
of an organic compound, which contains one or more hydroxyl groups.
For example, ethoxylated and/or propoxylated alcohol or ester
compounds or mixtures thereof are commonly available and are well
known to those skilled in the art. Suitable surfactants include,
but are not limited to, TYLOXAPOL; POLOXAMER 4070; POLOXAMER 188;
POLYOXYL 40 Stearate; POLYSORBATE 80, and POLYSORBATE 20, as well
as various compounds sold under the trade name TWEEN (ICI American
Inc., Wilmington, Del., U.S.A.), PLURONIC F-68 (trade name of BASF,
Ludwigshafen, Germany for a copolymer of polyoxyethylene and
polyoxypropylene). Preferred surfactants also include
polyoxyethylated oils or poloxamines. The TYLOXAPOL and TWEEN
surfactants are most preferred because they are FDA approved for
human use.
[0086] The parenteral nutrition emulsion composition can further
comprise carbohydrate nutrients such as, for example, dextrose;
electrolytes such as, for example, potassium and sodium chloride;
amino acids including essential and non-essential amino acids;
vitamins such as, for example, vitamin A, and vitamin D; trace
minerals such as, for example, zinc ions; and a preservative such
as, for example, methyl-, ethyl-, propyl-, and butylparaben, which
are medically accepted for parenteral administration.
[0087] The parenteral nutrition emulsion composition can further
comprise an osmolality modifier such as glycerin, sorbitol, or
alanine (see, for example, U.S. Pat. No. 4,567,045, the content of
which is incorporated by reference as if fully set forth herein),
and sterile water.
[0088] Generally, lipid droplets in emulsions for medical use
should preferably be small, i.e., below about 1 .mu.m, since the
smaller the droplets, the more stable the emulsion is in storage.
The droplet size is advantageously in the size range of about 0.05
to 0.5 .mu.m, and preferably about 0.1 to 0.3 .mu.m. The droplet
size is of particular importance since large droplets will not
readily pass through small blood capillaries and will not pass
through a filter required for filtration of the emulsion before its
administration to a subject in need thereof. The compositions of
the invention are particularly suitable for obtaining such small
droplets.
[0089] The emulsions of the present invention can be prepared by a
number of ways. In accordance with one preparation method, an
aqueous solution and an oily solution are separately prepared, the
aqueous solution comprising the phospholipids and optionally also
an osmotic pressure regulator and a preservative, and the oily
solution comprising the structured triglycerides, and an
antioxidant. The aqueous solution is prepared from two premade
solutions: a first, alcoholic solution containing the phospholipids
and a second solution containing the other optional ingredients
mentioned above in water. The aqueous solution is then prepared by
mixing the first and the second solutions, and removing the
alcohol, for example by evaporation, to yield the aforementioned
aqueous solution.
[0090] The aqueous solution and the oily solution are then mixed
with one another. However, the so-obtained mixture does not yet
consist of sufficiently small droplets, the size of which (obtained
after mixing with a magnetic stirrer) is about 10 .mu.m. The
droplet size of the inventive emulsion can then be decreased by the
use of emulsification equipment such as UltraTurrax (Jankl and
Kunkel, Staufen, FRG), which yields droplets having an average
diameter of about 1.1 .mu.m, or of a high shear mixer, e.g.,
Polytron (Kinematica, Lucerne, Switzerland), which yields droplets
having an average diameter of about 0.65 .mu.m.
[0091] Especially small droplets can be obtained in the inventive
emulsions by utilizing a two-stage pressure homogenizer in which
the crude dispersion is forced under high pressure through the
angular space between a spring loaded valve and the valve seat, the
second stage being in tandem with the first so that the emulsion is
subjected to two very rapid dispersion processes. An example of
such an apparatus is the Gaulin Homogenizer (APV Gaulin, Hilversum,
The Netherlands). Use of such an apparatus in accordance with the
invention yields emulsions in which the droplets have an average
diameter of about 0.27 .mu.m with a relatively small deviation.
[0092] Even smaller droplets can be obtained when the
emulsification process combines the use of both a Polytron-type
high shear mixer followed by homogenization. The droplet size,
which is obtained in such a combination, is about 0.1-0.15 .mu.m.
These relatively small size droplets are preferred when the
emulsion is to be used for intravenous administration or when the
formulation is to be sterilized by filtration.
[0093] According to some embodiments, the emulsions of the present
application are prepared by applying a high shear mixer
(Ultraturrax) for 5 min at a mixing rate of 10,000 RPM. Emulsions
obtained after this stage are further treated with a microfluidizer
at a pressure typically in the range of 800-2600 bar for five
minutes at room temperature. Thereafter, the emulsions are cooled
to room temperature and the mean droplet size is lower than 0.2
.mu.m. Emulsions obtained after the aforementioned two-stage
process are sterilized by filtration using a membrane filter having
a pore size of less than 0.45 .mu.m, and preferably less than 0.2
.mu.m. The pH in the emulsion is adjusted to a physiologically
accepted pH, typically pH of 6 to 8, with 0.5M NaOH or 0.5 M HCl
solutions.
[0094] Another method for preparing the parenteral nutrition
emulsion compositions of the invention is by mixing together a
liposome mixture and an oily mixture, each one prepared separately
beforehand. The liposome mixture comprises all the ingredients,
which in the final composition do not form part of the oily phase,
namely the phospholipids, and also the optional osmotic pressure
regulator and the preservative. The preparation of the liposome
mixture from these ingredients can be carried out by means known in
the art.
[0095] The oily mixture comprises the structured triglycerides, and
also the anti-oxidant.
[0096] After the liposome mixture is mixed together with the oily
mixture, an emulsion is formed having relatively large droplets,
e.g., about 10 .mu.m, which is further processed in a similar
manner as described above in connection with the first preparation
method, until an emulsion having fine homogenous droplets is
obtained.
[0097] Typically, the emulsions of the present invention are
filtered through a membrane filter having a pore size of less than
0.45 .mu.m, preferably a membrane filter having a pore size of 0.2
.mu.m. The emulsions are further sterilized by heating up to
121.degree. C. for 15 min under inert gas atmosphere such as, for
example, nitrogen, while rotating the emulsion during autoclaving.
However, any method for sterilization as known in the art is
encompassed in the present invention.
[0098] Distillation of the structured triglycerides is performed by
heating and evaporating the structured triglycerides, preferably
under vacuum. Preferably, the structured triglycerides of the
present invention are subjected to molecular or short-path
distillation, which is performed at low temperature under very low
pressure, typically below 0.01 mm Hg. Preferably, the distillation
is conducted at a temperature of 120.degree. C. to 230.degree. C.
The pressure can vary from about 1.times.10.sup.-3 kPa to 0.533
kPa. This low pressure enables the separation of high molecular
weight compounds such as the structured triglycerides of the
invention. Free fatty acids can also be removed from the reaction
mixture by washing with a sodium hydroxide or an alkoxide solution,
preferably of about 0.5M. Alternatively or additionally, the free
fatty acid can also be removed from the reaction mixture by steam
stripping.
[0099] The emulsions of the present invention are packaged and
stored in hermetically sealed containers for long or short-term
storage. The additives to be included in the emulsions will depend
upon how long the emulsions are to be stored. Long-term storage is
acceptable for emulsions with aqueous phases containing sugar, the
amino acids and some electrolytes. Dextrose should not be included
in emulsions prepared for long-term storage.
Advantages of Different Classes of Fatty Acids
[0100] The group of medium chain fatty acids (MCFA) includes fatty
acids that consist of 6-12 carbon atoms. Their chemical and
physical structure makes the MCFA more soluble than long or very
long chain fatty acids (LCFA or VLCFA, respectively); the latter
terms denote fatty acid of 14-18 and 20-22 carbon atoms,
respectively. All the fatty acids in the MCFA group are saturated.
Being rapidly oxidized, MCFA are considered as a very good source
of energy.
[0101] Emulsions containing medium chain triglycerides (MCT) are
more stable than those containing pure long chain triglycerides
(LCT). As MCT enter the blood stream, lipases cleave the
triglycerides hydrolytically to glycerol and free fatty acids.
Since MCFA do not need carnitine to enter the mitochondria, the
bulk of FA released is immediately taken up by the tissues and
rapidly oxidized. This metabolic pathway enables MCFA to be
eliminated from the blood stream more quickly than LCFA, and as
such they do not increase blood triglyceride levels and they have
low tendency of incorporation into tissue lipids. MCFA are also
known to preserve body protein, to increase nitrogen retention, to
decrease gluconeogenesis, and to improve nitrogen balance. MCFA
are, therefore, used as a rapid energy source.
[0102] LCFA consist of 14-18 carbon atoms. They can be saturated,
monounsaturated or polyunsaturated. Long chain triglycerides are
transported in the blood as lipoproteins. Lipoprotein lipase and
hepatic lipase hydrolyze LCT to FA and glycerol. The clearance of
LCT from the blood is slower than MCT. LCFA enter the mitochondria
by carnitine. LCFA function as an energy source by beta oxidation,
as precursors of longer chain FA, and as a storage in adipose
tissues. The most important LCFA are linoleic acid (18:2 .omega.-6)
and alpha linolenic (18:3 .omega.-3), both considered essential FA
since they cannot be synthesized by the human body, and therefore
must be provided in the diet.
[0103] The group of very long chain fatty acids (VLCFA) includes
chains of 20 carbon atoms or more. Most of the VLCFA are
polyunsaturated. They are synthesized from LCFA by elongation
process, which involves several enzymes. Among the most important
VLCFA are arachidonic acid (AA), an .omega.-6 fatty acid, and
docosahexaenoic (DHA), an .omega.-3 fatty acid, which have been
shown to be necessary for normal development and function of the
brain, the central nervous system (CNS) and the retina. AA and DHA
are not only mechanical components of the CNS structure, but also
required for cell signaling systems in neurons. There is evidence
linking DHA deficiency to attention deficit and hyperactivity
disorders, dyslexia, senile dementia, reduced visual and cognitive
function, clinical depression, schizophrenia and other problems of
psychological and physiological nature. In addition,
eicosapentaenoic acid (EPA) and DHA, both .omega.-3 fatty acids,
have been indicated to have beneficial effects on coronary heart
disease, hypertension, inflammation, arthritis, psoriasis, and
other autoimmune disorders and cancer. DHA and AA are also involved
in the synthesis of prostaglandins, thromboxanes and leukotrienes.
In addition, DHA and AA are crucial components of biological cell
membranes. It has been well established that fetus, preterm and
term infants require these fatty acids for their normal
development.
[0104] Incorporation rates of DHA and AA in red blood cell
membranes of infants were shown to decline without supplementation
of DHA and AA. Infants fed with human milk (which contains DHA and
AA) or formula supplemented with those fatty acids, were shown to
maintain normal rates of incorporation. Other studies show that
term and preterm infants fed with formula containing VLCFA exhibit
better cognitive behavior and psychomotor development than term and
preterm infants fed with formula that did not contain VLCFA.
[0105] The effect of .omega.-3 VLCFA, like DHA and EPA, on the
immune system has been studied in animals and humans. Surgical
patients that were given parenteral nutrition including DHA and EPA
showed a rise in interleukin 2 Patients having inflammatory bowel
diseases who received parenteral or enteral nutrition containing
DHA and EPA, showed an improvement in their clinical state with a
reduction of the steroid intake. These beneficial effects of VLCFA
.omega.-3 on inflammatory diseases is presumably due to their
involvement in interleukin production, which suppress inflammatory
processes.
[0106] The VLCFA .omega.-3 have also been shown to exert beneficial
effect on coronary heart diseases as they reduce platelet
aggregation and blood viscosity, increase capillary flow, and
reduce the risk of myocardial infarction. It should be appreciated
that the rate of conversion of these very long chain fatty acids
from their precursors is not adequate to fulfill the body
requirements, and therefore such fatty acids have to be included in
parenteral nutrition.
[0107] According to the principles of the present invention, at
least part of the triglycerides incorporated into the parenteral
nutrition emulsion comprise .omega.-6 fatty acids. It will be
appreciated that among the .omega.-6 VLCFA, AA is one of the more
preferred .omega.-6 fatty acids. According to the principles of the
present invention, although the structured triglycerides can
comprise any .omega.-6 fatty acid, gamma linolenic acid and
dihomogamma linolenic acid are not present simultaneously on the
same glycerol backbone with C.sub.18-22 n-3 fatty acid residue.
Vitamin E
[0108] Vitamin E is the nutritional designation of the tocopherols,
a group of essential biologically active substances. The various
tocopherols are found in germinal cells of plants, in egg yolk, and
in meat. The natural tocopherols include four isomers: alpha, beta,
gamma, and delta. The most biologically active vitamin E is the
alpha tocopherol isomer.
[0109] Consumption of fatty acids containing double bonds increases
the hazard of peroxide formation, which leads to structural changes
within cellular membranes. These changes are demonstrated
particularly in impairment of the immune system function, in
pulmonary complications, and in increased hemolysis.
[0110] Preterm infants and critically ill patients are more
vulnerable to peroxidation hazards. Vitamin E is a highly effective
antioxidant, protecting the double bonds of unsaturated fatty acids
from oxidative destruction. This protective function is
demonstrated both in vitro, in lipid emulsions, and in vivo, by
protecting the lipid fracture of membranes.
[0111] Vitamin E is also essential for the maintenance of a
functional immune system. In vitamin E deficiency there is a
decrease in the resistance to infection, in the immune response, in
the activation of T lymphocytes, in the production of interleukin
2, and in the phagocytic capacity. Patients receiving lipid
emulsion in parenteral nutrition have an increased requirement for
vitamin E.
[0112] The following examples are to be considered merely as
illustrative and non-limiting in nature. It will be apparent to one
skilled in the art to which the present invention pertains that
many modifications, permutations, and variations may be made
without departing from the scope of the invention.
Example 1
Identification of Lipases Suitable for Synthesis of Structured
Triglycerides
Materials and Methods
[0113] MCT solution was purchased from (Croda, Singapore). The MCT
solution contained 58% Caprylic acid and 42% Capric acid. According
to the manufacturer, both fatty acids are distributed randomly on
the glycerol backbone. Free fatty acids of different purities were
purchased from Sigma. DHA and EPA were obtained from K.D. Pharma,
Germany. Arachidonic acid bound to glycerol in a form of
triglycerides was obtained from Martek, USA, at a concentration of
20%. All solvents and chemicals were obtained from Sigma and were
of analytical grade. Lipases were purchased from Sigma, USA.
Acidolysis Reaction
[0114] The acidolysis activity of crude enzymes and enzymes adapted
for synthetic reactions was assayed by adding an enzyme preparation
(500 mg) into a 10 ml reaction solution containing MCT and a free
fatty acid where the molar ratio between both substrates was 1:1.
The reaction mixture was shaken for 16 hours at a temperature of
50.degree. C. Samples (0.1 ml) were taken from the reaction mixture
and mixed with 10 ml solvent comprised of acetone-dichloromethane
at a ratio of 90:10. The samples were filtered through a Millipore
filter (pore size 0.45 .mu.m) and then injected to the HPLC. The
reaction is presented in FIG. 1.
Analysis
[0115] The reaction progress was followed by analyzing the
triglyceride composition in the reaction medium before and after
the enzymatic reaction using an HPLC equipped with an ELSD
(Evaporative light Scattering Detector). The HPLC was equipped with
a LiChrosorb CH-18 Super (250.times.4 mm, 5 .mu.m, MERCK). The HPLC
running conditions were as follows: mobile phase A,
Acetonitrile/Dichloromethane/Acetone (80/15/5); mobile phase B:
Acetone/Dichloromethane/Acetone (20/60/20). The flow rate was 1
ml/min and the gradient was 0 to 100% B for 30 min.
[0116] FIGS. 2A and 2B show HPLC chromatograms before and after
acidolysis reaction of MCT. FIG. 2A, peak 1 represents the free
fatty acid, namely palmitic acid while peaks 2, 3, 4 and 5
represent the MCT before the reaction and peak 6 is trilaurin, an
internal standard. FIG. 2B, peaks 1, 2 and 3 represent the
liberated medium-chain fatty acids, while peak 4 represents the
non-reacted palmitic acid. Peaks 5, 6, 7 and 8 represent the
non-reacted MCT in the reaction medium, while peaks 9, 10, and 11
represent the new products containing one long-chain fatty acyl
group. Peaks 12 and 13 represent the new products containing two
long-chain fatty acyl groups.
[0117] The substrate conversion was calculated as follows:
Conversion %=100.times.(Sum of area of the 4 peaks representing the
MCT at time zero)-(Sum of area of the 4 peaks representing the MCT
after 16h reaction time)per Sum of area of the 4 peaks representing
the MCT at time zero.
The MCT conversion (%) was used as an indicative parameter for the
efficiency of enzyme activity to obtain the desired product.
Results
[0118] Tables 1-12 show the acidolysis activity of various lipases,
which were screened for their ability to catalyze the reaction
between MCT and free fatty acids in a solvent-free system according
to the enzymatically catalyzed reaction presented in FIG. 1. All
lipases were used in their crude form as supplied by the
manufacturer (Sigma) or immobilized on an insoluble matrix.
Immobilized lipases were activated before their use in the
acidolysis reaction containing MCT and a long-chain fatty acid by
pretreatment with a biphase system containing 50% triglyceride oil
such as olive oil, soy oil and the like and 50% water. This
pretreatment lead to the activation of the immobilized enzymes for
synthetic applications.
[0119] A typical enzyme activation and immobilization procedure
used in this example is as follows: 1 g of a crude lipase
preparation was dissolved in a 1 L of phosphate buffer solution of
an appropriate pH according to the recommendation of the enzyme's
manufacturer. 10 g of support matrix (silica, celite, an
ion-exchange resin such as DUOLITE.RTM. A56, DUOLITE.RTM. A7,
DUOLITE.RTM. XAD761, or Amberlite.TM. XAD16) were added into the
stirred enzyme solution. The slurry was shaken for 8 hours at a
temperature of 10.degree. C. Hundred ml of cold acetone was
optionally added into the stirred slurry in order to enhance the
enzyme immobilization on the matrix surface. The immobilized enzyme
was filtered of from the mixture and then freeze-dried or used wet
for the following step. One g of freeze-dried immobilized lipase
(water content of less than 2 weight %) or wet immobilized lipase
(water content of approx. 20%) was added into a biphase system
comprised of 5 g olive oil and 2 g buffer solution of pH7 at room
temperature. The mixture was stirred for 10 min. The immobilized
lipases was filtered of from the mixture, washed with cold acetone,
dried in a desiccator and then used for the acidolysis reaction
between MCT and a fatty acyl donor.
TABLE-US-00001 TABLE 1 The acidolysis activity of different enzymes
using MCT and caproic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 5 Candida Antarctica 30 Candida cylindracea 2
Mucor miehei 35 Pseudomonas fluorescens 4 Pseudomonas cepacia 27
Rhizopus arrhizus 42 Rhizopus niveus 3 Porcine pancreas lipase 6
Aspergillus oryzae 8 Candida lipolytica 8 Mucor javanicus 32
Penicillium roqueforti 3 Rhizomucor miehei 31 Wheat germ 2
Chromobacterium viscosum 18 Lipoprotein lipase Pseudomonas A 17
Lipoprotein lipase Pseudomonas B 18
[0120] Reaction conditions: MCT (1 gr), caproic acid (0.23 gr), and
500 mg enzyme preparation. The reaction mixture was shaken and
thermostated at 50.degree. C. for 16 h.
[0121] The results presented in Table 1 show that there are 6
different preferred sources for lipases for the acidolysis reaction
of Caproic acid and MCT. These sources of lipases include Candida
Antarctica, Mucor miehei, Pseudomonas cepacia, Rhizopus arrhizus,
Mucor javanicus and Rhizomucor miehei. Other lipases extracted form
different sources of microorganisms did not show adequate
acidolysis activity in organic medium.
TABLE-US-00002 TABLE 2 The acidolysis activity of different enzymes
using MCT and Caprylic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 3 Candida Antarctica 34 Candida cylindracea 5
Mucor miehei 42 Pseudomonas fluorescens 6 Pseudomonas cepacia 32
Rhizopus arrhizus 38 Rhizopus niveus 2 Porcine pancreas lipase 5
Aspergillus oryzae 9 Candida lipolytica 3 Mucor javanicus 37
Penicillium roqueforti 5 Rhizomucor miehei 37 Wheat germ 6
Chromobacterium viscosum 28 Lipoprotein lipase Pseudomonas A 15
Lipoprotein lipase Pseudomonas B 24
[0122] Reaction conditions were as described in Table 1, with one
modification, namely caprylic acid (0.28 gr) was used as the fatty
acid.
[0123] Table 2 shows that lipases extracted from Candida
Antarctica, Mucor miehei, Pseudomonas Cepacia, Rhizopus arrhizus,
Mucor javanicus, Rhizomucor miehei, Chromobacterium viscosum and
Lipoprotein lipase Pseudomonas A show reasonable acidolysis
activity using Caprylic acid and MCT as starting materials.
[0124] Tables 3-9 herein below also show that the most active
lipases to catalyze the reaction between MCT and FFA include mainly
the enzymes extracted from the aforementioned group of
microorganisms. These results indicate that such lipases are
efficient for acidolysis reactions where the substrates are MCT and
FFA providing that the fatty acid is saturated, mono-unsaturated,
di-unsaturated or tri-unsaturated and consists of 6-18 carbons.
TABLE-US-00003 TABLE 3 The acidolysis activity of different enzymes
using MCT and Capric acid. Lipase origin Conversion % (24 h)
Aspergillus niger 3 Candida Antarctica 25 Candida cylindracea 8
Mucor miehei 39 Pseudomonas fluorescens 9 Pseudomonas cepacia 37
Rhizopus arrhizus 42 Rhizopus niveus 3 Porcine pancreas lipase 5
Aspergillus oryzae 15 Candida lipolytica 5 Mucor javanicus 37
Penicillium roqueforti 5 Rhizomucor miehei 37 Wheat germ 6
Chromobacterium viscosum 28 Lipoprotein lipase Pseudomonas A 15
Lipoprotein lipase Pseudomonas B 24
[0125] Reaction conditions as described in Table 1, with one
modification, namely capric acid (0.34 gr) was used as the fatty
acid.
TABLE-US-00004 TABLE 4 The acidolysis activity of different enzymes
using MCT and Lauric acid. Lipase origin Conversion % (24 h)
Aspergillus niger 5 Candida Antarctica 14 Candida cylindracea 2
Mucor miehei 38 Pseudomonas fluorescens 12 Pseudomonas cepacia 17
Rhizopus arrhizus 29 Rhizopus niveus 5 Porcine pancreas lipase 6
Aspergillus oryzae 14 Candida lipolytica 8 Mucor javanicus 41
Penicillium roqueforti 7 Rhizomucor miehei 39 Wheat germ 4
Chromobacterium viscosum 31 Lipoprotein lipase Pseudomonas A 17
Lipoprotein lipase Pseudomonas B 27
[0126] Reaction conditions as described in Table 1, with one
modification, namely lauric acid (0.4 gr) was used as the fatty
acid.
TABLE-US-00005 TABLE 5 The acidolysis activity of different enzymes
using MCT and Palmitic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 3 Candida Antarctica 22 Candida cylindracea 6
Mucor miehei 41 Pseudomonas fluorescens 2 Pseudomonas cepacia 31
Rhizopus arrhizus 39 Rhizopus niveus 1 Porcine pancreas lipase 1
Aspergillus oryzae 15 Candida lipolytica 5 Mucor javanicus 40
Penicillium roqueforti 2 Rhizomucor miehei 33 Wheat germ 2
Chromobacterium viscosum 31 Lipoprotein lipase Pseudomonas A 15
Lipoprotein lipase Pseudomonas B 26
[0127] Reaction conditions as described in Table 1, with one
modification, namely palmitic acid (0.51 gr) was used as the fatty
acid.
TABLE-US-00006 TABLE 6 The acidolysis activity of different enzymes
using MCT and Stearic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 3 Candida Antarctica 34 Candida cylindracea 5
Mucor miehei 42 Pseudomonas fluorescens 6 Pseudomonas cepacia 32
Rhizopus arrhizus 38 Rhizopus niveus 2 Porcine pancreas lipase 5
Aspergillus oryzae 9 Candida lipolytica 3 Mucor javanicus 37
Penicillium roqueforti 5 Rhizomucor miehei 37 Wheat germ 6
Chromobacterium viscosum 28 Lipoprotein lipase Pseudomonas A 15
Lipoprotein lipase Pseudomonas B 30
[0128] Reaction conditions as described in Table 1, with one
modification, namely stearic acid (0.56 gr) was used as the fatty
acid.
TABLE-US-00007 TABLE 7 The acidolysis activity of different enzymes
using MCT and Oleic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 1 Candida Antarctica 20 Candida cylindracea 3
Mucor miehei 40 Pseudomonas fluorescens 3 Pseudomonas cepacia 37
Rhizopus arrhizus 41 Rhizopus niveus 1 Porcine pancreas lipase 4
Aspergillus oryzae 12 Candida lipolytica 1 Mucor javanicus 42
Penicillium roqueforti 2 Rhizomucor miehei 43 Wheat germ 2
Chromobacterium viscosum 33 Lipoprotein lipase Pseudomonas A 17
Lipoprotein lipase Pseudomonas B 29
[0129] Reaction conditions as described in Table 1, with one
modification, namely, oleic acid (0.56 gr) was used as the fatty
acid.
TABLE-US-00008 TABLE 8 The acidolysis activity of different enzymes
using MCT and Linoleic acid. Lipase origin Conversion % (24 h)
Aspergillus niger 2 Candida Antarctica 19 Candida cylindracea 2
Mucor miehei 41 Pseudomonas fluorescens 3 Pseudomonas cepacia 37
Rhizopus arrhizus 42 Rhizopus niveus 4 Porcine pancreas lipase 5
Aspergillus oryzae 17 Candida lipolytica 2 Mucor javanicus 33
Penicillium roqueforti 3 Rhizomucor miehei 35 Wheat germ 4
Chromobacterium viscosum 20 Lipoprotein lipase Pseudomonas A 12
Lipoprotein lipase Pseudomonas B 17
[0130] Reaction conditions as described in Table 1, with one
modification, namely linoleic acid (0.55 gr) was used as the fatty
acid.
TABLE-US-00009 TABLE 9 The acidolysis activity of different enzymes
using MCT and alpha-Linolenic acid. Lipase origin Conversion % (24
h) Aspergillus niger 1 Candida Antarctica 15 Candida cylindracea 2
Mucor miehei 31 Pseudomonas fluorescens 2 Pseudomonas cepacia 35
Rhizopus arrhizus 37 Rhizopus niveus 1 Porcine pancreas lipase 6
Aspergillus oryzae 12 Candida lipolytica 2 Mucor javanicus 22
Penicillium roqueforti 3 Rhizomucor miehei 39 Wheat germ 3
Chromobacterium viscosum 15 Lipoprotein lipase Pseudomonas A 13
Lipoprotein lipase Pseudomonas B 12
[0131] Reaction conditions as described in Table 1, with one
modification, namely alpha-linolenic acid (0.55 gr) was used as the
fatty acid.
TABLE-US-00010 TABLE 10 The acidolysis activity of different
enzymes using MCT and Arachidonic acid. Lipase origin Conversion %
(24 h) Aspergillus niger 3 Candida Antarctica 10 Candida
cylindracea 3 Mucor miehei 3 Pseudomonas fluorescens 6 Pseudomonas
cepacia 19 Rhizopus arrhizus 2 Rhizopus niveus 5 Porcine pancreas
lipase 2 Aspergillus oryzae 6 Candida lipolytica 2 Mucor javanicus
6 Penicillium roqueforti 4 Rhizomucor miehei 15 Wheat germ 2
Chromobacterium viscosum 12 Lipoprotein lipase Pseudomonas A 7
Lipoprotein lipase Pseudomonas B 8
[0132] Reaction conditions as described in Table 1, with one
modification, namely arachidonic acid (0.6 gr) was used as the
fatty acid.
[0133] The results presented in Table 10 show that most lipases
from the various sources did not catalyze the acidolysis of
arachidonic acid and MCT as efficiently as in the other experiments
disclosed hereinabove. This low acidolysis activity for the
different lipases is common in circumstances where the fatty acid
substrate contains high degree of unsaturation such that present in
ARA, EPA and DHA. The results in Table 10 show that lipases
extracted from Pseudomonas cepacia, Rhizomucor miehei, Candida
antarctica and Chromobacterium viscosum are among the most
efficient enzymes for the incorporation of ARA into MCT substrates.
Lipases extracted from Pseudomonas cepacia and Chromobacterium
viscosum are not certified for food applications, and therefore,
the other two lipases are being used for acidolysis of ARA and
MCT.
TABLE-US-00011 TABLE 11 The acidolysis activity of different
enzymes using MCT and EPA. Lipase origin Conversion % (24 h)
Aspergillus niger 2 Candida Antarctica 22 Candida cylindracea 3
Mucor miehei 25 Pseudomonas fluorescens 3 Pseudomonas cepacia 25
Rhizopus arrhizus 22 Rhizopus niveus 2 Porcine pancreas lipase 3
Aspergillus oryzae 14 Candida lipolytica 3 Mucor javanicus 29
Penicillium roqueforti 7 Rhizomucor miehei 30 Wheat germ 3
Chromobacterium viscosum 25 Lipoprotein lipase Pseudomonas A 6
Lipoprotein lipase Pseudomonas B 14
[0134] Reaction conditions as described in Table 1, with one
modification, namely EPA (0.6 gr) was used as the fatty acid.
TABLE-US-00012 TABLE 12 The acidolysis activity of different
enzymes using MCT and DHA. Lipase origin Conversion % (24 h)
Aspergillus niger 2 Candida Antarctica 24 Candida cylindracea 2
Mucor miehei 27 Pseudomonas fluorescens 3 Pseudomonas cepacia 19
Rhizopus arrhizus 21 Rhizopus niveus 3 Porcine pancreas lipase 5
Aspergillus oryzae 2 Candida lipolytica 3 Mucor javanicus 2
Penicillium roqueforti 5 Rhizomucor miehei 16 Wheat germ 6
Chromobacterium viscosum 12 Lipoprotein lipase Pseudomonas A 11
Lipoprotein lipase Pseudomonas B 10
[0135] Reaction conditions as described in Table 1, with one
modification, namely DHA (0.65 gr) was used as the fatty acid.
[0136] Tables 11 and 12 show that some lipases exhibit reliable
acidolysis activity for EPA and DHA when both were separately
interesterified with MCT. The results presented in Tables 11 and 12
show that lipases extracted from Candida Antarctica, Mucor miehei,
Pseudomonas Cepacia, Rhizopus arrhizus, and Rhizomucor miehei are
the most efficient enzymes for incorporation of DHA and EPA into
MCT.
[0137] These results demonstrate that long-chain fatty acyl groups
of different types can be efficiently incorporated in the sn-1
position of MCT molecules by using different lipases adapted to
applications in organic media. The most active lipases that are
certified for food applications and exchange medium-chain fatty
acyl groups bound on the sn-1 position of MCT molecules with
long-chain fatty acyl groups were identified and include those
extracted from Candida antarctica, Mucor miehei, Rhizopus arrhizus,
Mucor javanicus, Rhizomucor miehei, Thermomyces lanuginose and
Lipoprotein lipase Pseudomonas A. The present results also
demonstrate that the incorporation of polyunsaturated FAs (PUFAs)
in MCT molecules is also possible using the adapted reaction system
with adapted enzymes for organic synthesis.
Example 2
Synthesis of Structured Triglycerides
[0138] The acidolysis or inter-esterification reaction is initiated
by adding 1 g immobilized lipase preparation to 10 g MCT and an
equimolar amount of a free fatty acid or fatty acid ethyl ester.
The reaction mixture is shaken at 50.degree. C. for 2 to 8 hours.
The immobilized enzyme is used repeatedly, i.e., the reaction is
continued by leaving the immobilized enzyme in the reaction vessel
after completion of the reaction, and replacing the reaction
mixture with a freshly prepared reaction mixture comprising an MCT
and a fatty acid or fatty acid ethyl ester. Thereafter, the
triglyceride mixture is isolated using a molecular distillation
system where the first distilled fraction at a temperature of
120.degree. C. and a vacuum of 0.005 mmHg contains mainly the
medium- and long-chain free fatty acids or their ethyl esters and
partly the non-reacted MCT. The excess MCT in the medium is
distilled at 200.degree. C. and a pressure of 0.005 mmHg. The
resulting oil contains mainly reacted MCT with one long-chain fatty
acid at the sn-1 position (up to 80%) and also contains reacted MCT
with two long-chain fatty acyl groups at the sn-1 and sn-3
positions. To protect the oil from oxidation, alpha tocopherol is
added (2 mg per 1 g of structured oil) into the structured oil.
This oil is sterilized prior to emulsification by filtration
through a sterilizing filter (pore size 0.2 .mu.m).
Example 3
Preparation of Structure Triglycerides of Predetermined Content of
Fatty Acids
[0139] The starting material MCTs composed of 58.3% C8 and 41.7%
C10 (100 g, 0.2 mol) was mixed with a donor of a specific
long-chain fatty acid, preferably a free fatty acid or ethyl fatty
acid ester (0.24 mol). The solution was heated to 55.degree. C. to
obtain a homogenous mixture. A lipase (10 g) of 1,3-positional
specificity, such as Lipozyme RM IM or any other lipase preparation
was added to the reaction mixture. The reaction mixture was shaken
at 55.degree. C. for 8 hours. The enzyme was filtered off from the
reaction medium for repeated use. The filtrate was subjected to
molecular distillation where the first fraction was collected at a
temperature of 120.degree. C. and a pressure of 0.005 mmHg, which
contained mainly the non-reacted fatty acids and the medium-chain
fatty acids produced in the reaction. The second fraction was
collected at a temperature of 200.degree. C. and a pressure of
0.005 mmHg, which contained mainly the non-reacted MCTs. The
residue of the distilled reaction mixture (approximately 90 g)
mainly contained the MCTs predominantly monoacylated at the sn-1
position with the specific long-chain fatty acid (80-90%) and the
MCTs predominantly diacylated at the sn-1 and sn-3 position
(10-20%) with the specific long-chain fatty acid present in the
starting material. Different proportions of reaction residues were
mixed together to obtain the desired structured triglycerides
composition with regard to the type, concentration and position on
the glycerol backbone for the attached fatty acid. Alpha-tocopherol
(2 mg/1 g of oil) was added to the structured triglycerides mixture
prior to emulsification.
Example 4
Preparation of Structured Triglyceride Mixture Containing
Arachidonic Acid
[0140] MCTs (100 g, 0.2 mol) and either Arachidonic acid (0.24 mol,
free acid or its ethyl ester) or Martek's oil containing ARA and
DHA each approximately 20% (50 g) were mixed together to obtain a
homogenous solution. A lipase (10 g) of 1,3-positional specificity,
such as Lipozyme RM IM or any other lipase preparation was added to
the reaction mixture. The reaction mixture was shaken at 50.degree.
C. for 8 hours. The enzyme was filtered off from the reaction
medium for repeated use. The filtrate was subjected to molecular
distillation where the first fraction was collected at a
temperature of 120.degree. C. and a pressure of 0.005 mmHg, which
contained mainly the unreacted fatty acids or their ethyl esters
and the medium-chain fatty acids produced in the reaction. The
second fraction was collected at a temperature of 200.degree. C.
and a pressure of 0.005 mm Hg, which contained mainly the
non-reacted MCTs. The residue (approximately 90 g) mainly contained
the MCTs predominantly monoacylated at the sn-1 position with the
specific long-chain fatty acid (80-90%) and the MCTs predominantly
diacylated at the sn-1 and sn-3 position (10-20%) with the specific
long-chain fatty acid present in the starting material.
Example 5
Preparation of an Emulsion Comprising the Structured
Triglycerides
[0141] 200 g of the structured triglyceride preparation were
prepared in a large volume according to the procedure disclosed
hereinabove (Example 2). The structured triglycerides preparation
was added to a homogenized mixture comprised of 22.5 g glycerol, 12
g phospholipid (Lipoid E 80) and 765.5 g distilled water adjusted
to a desired pH value. The addition of sodium oleate (0.3 g) as an
emulsion stabilizer into the mixture was optional. The mixture was
homogenized several times with a homogenizer (Ultraturrax) at a
rate of 10,000 rpm. In all steps adopted for preparation of the
emulsion the temperature did not exceed 70.degree. C. The obtained
macro-emulsion was treated further in a high-pressure homogenizer
(Microfluidizer) at 2600 psi for 5 minutes at temperature below
40.degree. C. After this treatment the emulsion was passed through
a membrane filter of pore size of 0.2 .mu.m.
[0142] The composition of the parenteral nutrition is thus as
follows: [0143] Structured triglycerides--20% (w/v) [0144] Alpha
tocopherol--1.8 mg/1 g fatty acids [0145] Phospholipids--12 g/liter
[0146] Glycerin--25 g/L [0147] Water to complete to 1 liter.
[0148] After filling 200 ml aliquots of said lipid emulsion into
plastic bags, the plastic bags are sterilized using high-pressure
steam for 20 minutes at 121.degree. C. to obtain a nutrition
emulsion composition.
Example 6
Preparation of a Structured Triglyceride Composition
[0149] This example illustrates the fatty acid composition of
structured triglycerides. As shown in Table 13, MCFA, LCFA, and
VLCFA constitute 40-50%, 35-55%, and 4.5-5.5% by weight,
respectively, of total FA in the structured triglycerides. The
ratio of .omega.-6 to .omega.-3 fatty acids in the structured
triglycerides is 1.75.
TABLE-US-00013 TABLE 13 Fatty acid composition of structured
triglycerides (% by weight). Caproic acid 6:0 0-5 Caprylic acid 8:0
20-30 Capric acid 10:0 10-30 Lauric acid 12:0 0-5 Myristic acid
14:0 0-5 Palmitic acid 16:0 5-30 Palmitoleic acid 16:1 0-5 Stearic
acid 18:0 0-5 Oleic acid 18:1 10-30 Linoleic acid 18:2 .omega.-6
10-30 Alpha linolenic acid 18:3 .omega.-3 5-15 Arachidonic acid
(AA) 20:4 .omega.-6 1-5 Ecosapentaenoic acid (EPA) 20:5 .omega.-3
0-5 Docosahexaenoic acid (DHA) 22:6 .omega.-3 1-5
Example 7
Preparation of a Structured Triglyceride Composition
[0150] This example illustrates the fatty acid composition of
structured triglycerides. As shown in Table 14, MCFA, LCFA, and
VLCFA constitute 45, 50.5, and 4.5% by weight, respectively, of
total FA in the structured triglycerides. The ratio of .omega.-6 to
.omega.-3 fatty acids in the structured triglycerides is 1.75.
TABLE-US-00014 TABLE 14 Fatty acid composition of structured
triglycerides (%). Caproic acid 6:0 2.5 Caprylic acid 8:0 30 Capric
acid 10:0 10 Lauric acid 12:0 2.5 Palmitic acid 16:0 10 Stearic
acid 18:0 2.5 Oleic acid 18:1 15 Linoleic acid 18:2 .omega.-6 16
Alpha linolenic acid 18:3 .omega.-3 7 Arachidonic acid (AA) 20:4
.omega.-6 1.5 Ecosapentaenoic acid (EPA) 20:5 .omega.-3 1.5
Docosahexaenoic acid (DHA) 22:6 .omega.-3 1.5
[0151] It will be appreciated by persons skilled in the art that
the present invention is not limited by what has been particularly
shown and described herein above. Rather the scope of the invention
is defined by the claims that follow.
* * * * *