U.S. patent application number 11/996016 was filed with the patent office on 2008-11-06 for compositions and methods for making and using nanoemulsions.
This patent application is currently assigned to University of Massachusetts Lowell. Invention is credited to Robert Nicolosi, Thomas Wilson.
Application Number | 20080274195 11/996016 |
Document ID | / |
Family ID | 38957224 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274195 |
Kind Code |
A1 |
Nicolosi; Robert ; et
al. |
November 6, 2008 |
Compositions and Methods for Making and Using Nanoemulsions
Abstract
The present invention discloses an improved nanoemulsion
comprising a uniform and discrete range of very small particle
nano-sized diameters. This uniformity results in improved
bioavailability of incorporated compounds (i.e., pharmaceuticals or
nutraceuticals) as reflected in various pharmacokinetic parameters
including, but not limited to, decreased T.sub.max, increased
C.sub.max, and increased AUC. The improved method of making these
uniform nanoemulsions utilizes microfluidization which differs in
both process and mechanics when compared to conventional milling
and grinding techniques used to generate nanoparticulate
compositions. Further, the improvement results, in part, from a
novel step of mixing a substantially soluble compound into a heated
dispersion medium. This is unlike current nanoparticulate
composition methods that mix an insoluble compound with an unheated
dispersion medium. Further, these nanoemulsions are observed to be
bacterial-resistant and stable to extremes in both temperature and
pH changes. Consequently, these nanoemulsions are expected to have
a significantly prolonged shelf-life than currently available
nanoemulsions.
Inventors: |
Nicolosi; Robert; (Nashua,
NH) ; Wilson; Thomas; (Bradford, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
University of Massachusetts
Lowell
Lowell
MA
|
Family ID: |
38957224 |
Appl. No.: |
11/996016 |
Filed: |
July 11, 2006 |
PCT Filed: |
July 11, 2006 |
PCT NO: |
PCT/US06/26918 |
371 Date: |
February 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60700224 |
Jul 18, 2005 |
|
|
|
Current U.S.
Class: |
424/489 ;
424/555; 514/169; 514/458; 514/729; 977/906 |
Current CPC
Class: |
A61K 9/1075 20130101;
A61P 3/06 20180101; B01F 2003/0849 20130101; Y10S 977/906 20130101;
Y10S 977/773 20130101 |
Class at
Publication: |
424/489 ;
514/169; 424/555; 514/458; 514/729; 977/906 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/01 20060101 A61K031/01; A01N 25/00 20060101
A01N025/00; A61K 31/56 20060101 A61K031/56; A61K 35/60 20060101
A61K035/60; A61K 31/355 20060101 A61K031/355; A61K 31/047 20060101
A61K031/047; A61K 38/16 20060101 A61K038/16; A01P 1/00 20060101
A01P001/00 |
Claims
1. A nanoemulsion comprising a population of particles having
diameters between approximately 10 and approximately 110
nanometers, wherein said nanoemulsion is not contaminated by
particles having diameters larger than 110 nanometers.
2. The nanoemulsion of claim 1, wherein said particles encapsulate
a compound.
3. The nanoemulsion of claim 2, wherein said compound is a
pharmaceutical.
4. The nanoemulsion of claim 2, wherein said compound is a
nutraceutical.
5. A nanoemulsion comprising a first and second population of
particles, wherein the majority of particles in said first
population have diameters between approximately 10 and
approximately 20 nanometers, wherein the majority of particles in
said second population have diameters between approximately 40 and
approximately 80 nanometers, wherein said nanoemulsion is
uncontaminated by particles having diameters larger than 110
nanometers.
6. The nanoemulsion of claim 5, wherein said particles encapsulate
a compound.
7. The nanoemulsion of claim 6, wherein said compound is a
pharmaceutical.
8. The nanoemulsion of claim 6, wherein said compound is a
nutraceutical.
9. A method, comprising: a) providing; i) a premix comprising a
compound and a liquid dispersion medium, wherein said compound has
a solubility greater than 30 mg/ml in said medium; and ii) a
microfluidizer capable of maintaining at least 25,000 PSI; b) using
a single pass exposure of said premix to said microfluidizer to
create a population of nanoemulsion particles having diameters
ranging approximately between 10-110 nm.
10. The method of claim 9, wherein said dispersion medium is
selected from the group consisting of aqueous media and oil-based
media.
11. The method of claim 10, wherein said aqueous media is selected
from the group consisting of water, saline solution, ringers
solution, dextrose, and short chain alcohols.
12. The method of claim 10, wherein said oil-based media is
selected from the group consisting of saturated and unsaturated
oils from vegetable and marine sources, silicone oils, and mineral
oils.
13. The method of claim 9, wherein said compound is selected from
the group consisting of a plant sterol, cod liver oil, tocopherol,
lecithin, lutein, zeaxanthin, and soy protein.
14. A method, comprising: a) providing; i) a premix comprising a
compound, a first antioxidant, a second antioxidant, and an aqueous
dispersion medium, wherein said compound has a solubility greater
than 30 mg/ml in said medium; and iii) a microfluidizer capable of
maintaining at least 25,000 PSI; c) using a single pass exposure of
said premix to said microfluidizer to create a population of
nanoemulsion particles having diameters ranging from between
approximately 40-110 nm, wherein said particle diameter remains
stable for at least four months.
15. The method of claim 14, further comprising pasteurizing said
population of nanoemulsion particles wherein said particle
diameters remain stable.
16. The method of claim 14, further comprising freezing said
population of nanoemulsion particles wherein said particle
diameters remain stable.
17. A method, comprising; a) providing; i) a subject refractory to
an administered compound at a therapeutically effective amount; ii)
a nanoemulsion comprising a population of particles encapsulating
said compound, wherein said particles having diameters between
approximately 10 and approximately 110 nanometers, wherein said
nanoemulsion is not contaminated by particles having diameters
larger than 110 nanometers; b) delivering said nanoemulsion to said
subjects under conditions such that said compound bioavailability
is improved and wherein said compound is therapeutically
effective.
18. The method of claim 17, wherein said improved bioavailability
comprises pharmacokinetic parameters selected from the group
consisting of decreased T.sub.max, increased C.sub.max, and
increased AUC.
19. The method of claim 17, wherein said delivering comprises a
method selected from the group consisting of oral, transdermal,
intravenous, intraperitoneal, intramuscular, and subcutaneous.
20. The method of claim 17, wherein said nanoemulsion comprises a
plant sterol.
21. The method of claim 17, wherein said nanoemulsion comprises
lycopene.
22. A nanoemulsion having bacteria-resistant properties, wherein
said nanoemulsion comprises a population of particles encapsulating
said compound, wherein said particles having diameters between
approximately 10 and approximately 110 nanometers, wherein said
nanoemulsion is not contaminated by particles having diameters
larger than 110 nanometers.
23. The nanoemulsion of claim 22, wherein said nanoemulsion resists
bacterial growth for at least three months.
24. The nanoemulsion of claim 22, wherein said bacterial-resistant
properties comprise shear-force induced cell lysis.
25. The nanoemulsion of claim 22, wherein said bacterial-resistant
properties comprise an oxidizing environment.
26. The nanoemulsion of claim 22, wherein said nanoemulsion is
sterile.
27. A method, comprising: a) providing; i) a premix comprising a
compound and a liquid dispersion medium; and ii) a device capable
of creating a continuous turbulent flow under high pressure; b)
using said device to create a population of nanoemulsion particles
having uniform diameter.
28. The method of claim 27, wherein said dispersion medium is
selected from the group consisting of aqueous media and oil-based
media.
29. The method of claim 28, wherein said aqueous media is selected
from the group consisting of water, saline solution, ringers
solution, dextrose, and short chain alcohols.
30. The method of claim 28, wherein said oil-based media is
selected from the group consisting of saturated and unsaturated
oils from vegetable and marine sources, silicone oils, and mineral
oils.
31. The method of claim 27, wherein said compound is selected from
the group consisting of a plant sterol, cod liver oil, tocopherol,
lecithin, lutein, zeaxanthin, and soy protein.
Description
FIELD OF INVENTION
[0001] The present invention relates to the field of nanoemulsions.
In one embodiment, nanoemulsions are made using high shear stress
technology. In one embodiment, the invention comprises uniform
microfluidized nanoemulsions. In another embodiment, the uniform
nanoemulsion comprises a compound such as a pharmaceutical,
nutraceutical, or cosmeceutical. In one embodiment, the uniform
nanoemulsion comprises improved pharmacokinetic parameters when
compared to conventional nanoparticulate compositions and/or
nanoemulsions. In one embodiment, the present invention
contemplates a method of making a bacteria-resistant
nanoemulsion.
BACKGROUND OF THE INVENTION
[0002] Micro/nanoemulsion technology has substantial commercial
value. In relation to the nutraceutical area alone, the market
value is estimated as a 250 billion dollar business world-wide.
Consequently, the ability to incorporate lipid soluble
nutraceuticals into beverages (the fastest-growing component of the
food industry) as well as low or no fat foods is of important
interest.
[0003] What is needed is a nanoemulsion that: i) has improved
temperature and pH stability; ii) improved bioavailability; and
iii) improved shelf-life due to microbial resistance. In addition,
nanoemulsions should be relatively easy and inexpensive to
prepare.
SUMMARY
[0004] The present invention relates to the field of nanoemulsions.
In one embodiment, the nanoemulsion is made using a high shear
stress technology. In one embodiment, the invention comprises
uniform microfluidized nanoemulsions. In another embodiment, the
uniform nanoemulsion comprises a compound such as a pharmaceutical,
nutraceutical, or cosmeceutical. In one embodiment, the uniform
nanoemulsion comprises improved pharmacokinetic parameters when
compared to conventional nanoparticulate compositions and/or
nanoemulsions. In one embodiment, the present invention
contemplates a method of making a bacteria-resistant
nanoemulsion.
[0005] In one embodiment, the present invention contemplates a
nanoemulsion comprising a population of particles having maximum
and minimum diameters, wherein the difference between said maximum
and minimum diameters does not exceed 100 nm.
[0006] In one embodiment, the present invention contemplates a
nanoemulsion comprising a population of particles having diameters
between approximately 10 and approximately 110 nanometers, wherein
said nanoemulsion is not contaminated by particles having diameters
larger than 110 nanometers. In one embodiment, the particles
encapsulate a compound. In one embodiment, the compound is a
pharmaceutical. In another embodiment, the compound is a
nutraceutical.
[0007] In one embodiment, the present invention contemplates a
nanoemulsion comprising a first and second population of particles,
wherein the majority of particles in said first population have
diameters between approximately 10 and approximately 20 nanometers,
wherein the majority of particles in said second population have
diameters between approximately 40 and approximately 80 nanometers,
wherein said nanoemulsion is uncontaminated by particles having
diameters larger than 110 nanometers. In one embodiment, the
particles encapsulate a compound. In one embodiment, the compound
is a pharmaceutical. In one embodiment, the compound is a
nutraceutical. A nanoemulsion comprising a population of particles
having diameters between approximately 50 and approximately 150
nanometers, wherein said nanoemulsion is not contaminated by
particles having diameters larger than 160 nanometers. In one
embodiment, the particles encapsulate a compound. In one
embodiment, the compound is a pharmaceutical. In one embodiment,
the compound is a nutraceutical.
[0008] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a premix comprising a compound
and a liquid dispersion medium, wherein said compound has a
solubility greater than 30 mg/ml in said medium; and ii) a
microfluidizer capable of maintaining at least 25,000 PSI; b) using
a single pass exposure of said premix to said microfluidizer to
create a population of nanoemulsion particles having diameters
ranging approximately between 10-110 nm. In one embodiment, the
dispersion medium is selected from the group consisting of aqueous
media and oil-based media. In one embodiment, the aqueous media is
selected from the group consisting of water, ringers solution,
dextrose, and short chain alcohols. In one embodiment, the
oil-based media is selected from the group including, but not
limited to, saturated and unsaturated oils from vegetable and
marine sources, silicone oils, mineral oils, and plant-derived
oils. In one embodiment, the compound is selected from the group
including, but not limited to, a plant sterol, cod liver oil,
tocopherol, lecithin, lutein, zeaxanthin, and soy protein.
[0009] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a heated dispersion medium;
ii) a compound having substantial solubility in said medium; and
iii) a microfluidizer capable of making a uniform nanoemulsion from
said medium; b) adding said compound to said medium at a
temperature of at least 70.degree. C. to create a premix; and c)
microfluidizing said premix at a pressure of at least 25,000 PSI to
create said nanoemulsion having particle diameters ranging between
10-110 nm. In one embodiment, said dispersion medium is selected
from the group consisting of soybean oil and water. In one
embodiment, said dispersion medium is heated to at least 65.degree.
C. In one embodiment, said compound may be selected from the group
comprising a plant sterol, cod liver oil, tocopherol, lecithin,
lutein, zeaxanthin, lycopene, whey protein, and soy protein. In one
embodiment, the nanoemulsion encapsulates the compound. In one
embodiment, 86% of said particle diameters have a 54 nm average
diameter. In one embodiment, 14% of said particles diameters have a
16 nm average diameter. In one embodiment, 82% of said particle
diameters have a 64 nm average diameter. In one embodiment, 17% of
said particle diameters have a 19 nm average diameter. In one
embodiment, 78% of said particle diameters have a 88 nm average
diameter. In one embodiment, 22% of said particle diameters have a
27 nm average diameter. In one embodiment, 84% of said particle
diameters have a 90 nm average diameter. In one embodiment, 16% of
said particle diameters have a 23 nm average diameter. In one
embodiment, 80% of said particle diameters have a 55 nm average
diameter.
[0010] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a premix comprising a
compound, a first antioxidant, a second antioxidant, and an aqueous
dispersion medium, wherein said compound has a solubility greater
than 30 mg/ml in said medium; and iii) a microfluidizer capable of
maintaining at least 25,000 PSI; c) using a single pass exposure of
said premix to said microfluidizer to create a population of
nanoemulsion particles having diameters ranging from between
approximately 40-110 nm, wherein said particle diameter remains
stable for at least four months. In one embodiment, the method
further comprises pasteurizing said population of nanoemulsion
particles wherein said particle diameters remain stable. In one
embodiment, the method further comprises freezing said population
of nanoemulsion particles wherein said particle diameters remain
stable.
[0011] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a stable aqueous dispersion
medium comprising a first antioxidant; ii) a solution comprising
natural emulsifiers; ii) a compound having substantial solubility
in said medium comprising a second antioxidant; and iii) a
microfluidizer capable of making a uniform nanoemulsion from said
medium; b) adding said compound and said solution to said medium
and heating to a temperature of at least 50.degree. C. to create a
premix; and c) microfluidizing said premix at a pressure of at
least 25,000 PSI to create said nanoemulsion having particle
diameters ranging between 40-110 nm wherein said particle diameter
remains stable for at least four months. In one embodiment, the
nanoemulsion encapsulates the compound. In one embodiment, the
method further comprises pasteurizing said nanoemulsion wherein
said particle diameters remain stable. In one embodiment, the
method further comprises freezing said nanoemulsion wherein said
particle diameters remain stable. In one embodiment, said solution
comprises milk. In one embodiment, said compound comprises DHA fish
oil. In one embodiment, said pasteurization comprises exposing said
nanoemulsions to 75.degree. C. for thirty seconds. In one
embodiment, said freezing comprises exposing said nanoemulsions to
-4.degree. C. for 24 hours.
[0012] In one embodiment, the present invention contemplates a
method, comprising; a) providing; i) a subject refractory to an
administered compound at a therapeutically effective amount; ii) a
nanoemulsion comprising a population of particles encapsulating
said compound, wherein said particles having diameters between
approximately 10 and approximately 110 nanometers, wherein said
nanoemulsion is not contaminated by particles having diameters
larger than 110 nanometers; b) delivering said nanoemulsion to said
patients under conditions such that said compound bioavailability
is improved and wherein said compound is therapeutically effective.
In one embodiment, the improved bioavailability comprises
pharmacokinetic parameters selected from the group consisting of
decreased T.sub.max, increased C.sub.max, and increased AUC. In one
embodiment, the delivering comprises a method selected from the
group consisting of oral, transdermal, intravenous,
intraperitoneal, intramuscular, and subcutaneous. In one
embodiment, the nanoemulsion comprises a plant sterol. In one
embodiment, the nanoemulsion comprises lycopene.
[0013] In one embodiment, the present invention contemplates a
method for improving a nanoemulsion bioavailability comprising
providing a uniform microfluidized nanoemulsion and delivering the
uniform nanoemulsion to a subject. In one embodiment, the subject
comprises a mammal. In one embodiment, the nanoemulsion
encapsulates a compound. In one embodiment, the nanoemulsion is
delivered by oral administration. In another embodiment, the
nanoemulsion is delivered by methods including, but not limited to,
transdermally, intravenously, intraperitoneally, intramuscularly or
subcutaneously. In one embodiment, said improved bioavailability
comprises pharmacokinetic parameters selected from the group
consisting of decreased T.sub.max, increased C.sub.max, and
increased AUC. In one embodiment, said nanoemulsion is formulated
for oral administration. In one embodiment, said nanoemulsion
comprises a plant sterol. In one embodiment, said nanoemulsion
comprises lycopene.
[0014] In one embodiment, the present invention contemplates a
nanoemulsion having bacteria-resistant properties, wherein said
nanoemulsion comprises a population of particles encapsulating said
compound, wherein said particles having diameters between
approximately 10 and approximately 110 nanometers, wherein said
nanoemulsion is not contaminated by particles having diameters
larger than 110 nanometers. In one embodiment, the nanoemulsion
resists bacterial growth for at least three months. In one
embodiment, the bacterial-resistant properties comprise shear-force
induced cell lysis. In one embodiment, the bacterial-resistant
properties comprise an oxidizing environment. In one embodiment,
the nanoemulsion is sterile.
[0015] In one embodiment, the present invention contemplates a
uniform microfluidized nanoemulsion comprising bacteria-resistant
properties. In one embodiment, said nanoemulsion resists bacterial
growth for at least three months. In one embodiment, the
nanoemulsion comprises particles having a diameter distribution of
between 10-110 nm. In one embodiment, said bacterial-resistant
properties comprise shear-force induced cell lysis. In one
embodiment, the nanoemulsion is sterile.
[0016] In one embodiment, the present invention contemplates a
method, comprising: a) providing; i) a premix comprising a compound
and a liquid dispersion medium; and ii) a device capable of
creating a continuous turbulent flow under high pressure; b) using
said device to create a population of nanoemulsion particles having
uniform diameter. In one embodiment, the dispersion medium is
selected from the group consisting of aqueous media and oil-based
media. In one embodiment, the aqueous media is selected from the
group consisting of water, saline solution, ringers solution,
dextrose, and short chain alcohols. In one embodiment, the
oil-based media is selected from the group consisting of saturated
and unsaturated oils from vegetable and marine sources, silicone
oils, and mineral oils. In one embodiment, the compound is selected
from the group consisting of a plant sterol, cod liver oil,
tocopherol, lecithin, lutein, zeaxanthin, and soy protein.
DEFINITIONS
[0017] In general, the terms used herein are to be interpreted
according to definitions generally accepted by those having
ordinary skill in the art. Those listed below, however, are to be
interpreted according to the following definitions.
[0018] The term "microfluidized", "microfluidizing", or
"microfluidizer" as used herein refers to an instrument or a
process that utilizes a continuous turbulent flow at high pressure
including, but not limited to, a microfluidizer or other like
device that may be useful in creating a uniform nanoemulsion. For
example, microfluidizing may create a uniform nanoemulsion
comprising a pharmaceutical, nutraceutical, or cosmeceutical from a
premix within a thirty (30) second time frame (typically referred
to a single pass exposure). Typically, a microfluidizer may be
operated at a pressure of approximately 25,000 PSI to generate a
uniform nanoemulsion.
[0019] The term "uniform nanoemulsion" as used herein, refers to
any emulsion comprising any specified range of particle diameter
sizes wherein the difference between the minimum diameter and
maximum diameters do not exceed approximately 600 nm, preferably
approximately 300 nm, more preferably approximately 200 nm, but
most preferably approximately 100 nm (i.e., for example,
microfluidization, as contemplated herein, produces a uniform
nanoemulsion having a range of approximately 10-110 nm and is
referred to herein as a uniform microfluidized nanoemulsion).
Preferably, the total particle distribution (i.e., 100%) is
encompassed within the specified range of particle diameter size. A
particle diameter distribution where less than 3% is outside the
specified range of particle diameter sizes is still contemplated
herein as a uniform nanoemulsion.
[0020] The term "population" as used herein, refers to any mixture
of nanoemulsion particles having a distribution in diameter size.
For example, a population of nanoemulsion particles may range is
particle diameter from between approximately 10-10 nm.
[0021] The term "nanoparticle" as used herein, refers to any
particle having a diameter of less than 300 nanometers (nm), as
defined by the National Science Foundation or preferably less than
100 nm, as defined by the National Institutes of Health. Most
conventional techniques create nanoparticle compositions with an
average particle diameter of approximately 300 nanometers (nm) or
greater.
[0022] The term "dispersion medium" as used herein, refers to any
oil-based or aqueous liquid wherein a pharmaceutical,
nutraceutical, or cosmeceutical may be dissolved upon heating.
Oil-based liquids may include, but not limited to; saturated and
unsaturated oils from vegetable and marine sources including, but
not limited to, soybeans, safflowers, olives, corn, cottonseeds,
linseed, safflower, palm, peanuts, flaxseeds, sunflowers, rice
bran, sesame, rapeseed, cocoa butter etc., and mixtures thereof;
silicone oils; and mineral oils. Alternatively, aqueous media may
include, but are not limited to, water, saline solutions, short
chain alcohols, 5% dextrose, Ringer's solutions (lactated Ringer's
injection, lactated Ringer's plus 5% dextrose injection, acylated
Ringer's injection), Normosol-M, Isolyte E, and the like; and
synthetic and/or natural detergents having high surfactant
properties, deoxycholates, cyclodextrins, chaotropic salts and ion
pairing agents etc., and mixtures thereof.
[0023] The term "compound" as used herein, refers to any
pharmaceutical, nutraceutical, or cosmeceutical (i.e., for example,
organic chemicals, lipids, proteins, oils, vitamins, crystals,
minerals etc.) that are substantially soluble in a dispersion
medium.
[0024] The term "substantially soluble" as used herein, refers to
any compound that dissolves into a dispersion medium to a
concentration greater than 30 mg/ml. Preferably, the dispersion
medium is heated while the compound is being dissolved.
[0025] The term "premix" as used herein, refers to any mixture that
is subsequently used to generate a nanoparticulate composition or a
uniform microfluidized nanoemulsion. Typically, premixes contain a
liquid dispersion medium and a compound, and optionally, an
emulsifier and/or an antioxidant.
[0026] The term "stable" as used herein, refers to any population
of nanoemulsion particles whose diameters stay within the range of
approximately 10-110 nm over a prolonged period of time (i.e., for
example, one (1) day to twenty-four (24) months, preferably, two
(2) weeks to twelve (12) months, but more preferably two (2) months
to five (5) months). For example, if a population of nanoemulsion
particles is subjected to prolonged storage, temperature changes,
and/or pH changes whose diameters stay within a range of between
approximately 10-110 nm, the nanoemulsion is stable.
[0027] The term "bacteria-resistant" as used herein refers to the
lack of observable bacterial growth.
[0028] The term "sterile" as used herein refers to a nanoemulsion
that contains no living bacterial cells.
[0029] The term "pharmaceutically acceptable" as used herein,
refers to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0030] The term "pharmaceutically acceptable salts" as used herein,
refers to derivatives wherein the parent compound is modified by
making acid or base salts thereof. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the
like. The pharmaceutically acceptable salts include the
conventional non-toxic salts or the quaternary ammonium salts of
the parent compound formed, for example, from non-toxic inorganic
or organic acids. For example, such conventional non-toxic salts
include those derived from inorganic acids such as hydrochloric,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isethionic, and the like.
[0031] The term "therapeutically effective amount" as used herein,
with respect to a drug dosage, shall mean that dosage that provides
the specific pharmacological response for which the drug is
administered or delivered to a significant number of subjects in
need of such treatment. It is emphasized that `therapeutically
effective amount,` administered to a particular subject in a
particular instance will not always be effective in treating the
diseases described herein, even though such dosage is deemed a
"therapeutically effective amount" by those skilled in the art.
Specific subjects may, in fact, be "refractory" to a
"therapeutically effective amount". For example, a refractory
subject may have a low bioavailability such that clinical efficacy
is not obtainable. It is to be further understood that drug dosages
are, in particular instances, measured as oral dosages, or with
reference to drug levels as measured in blood.
[0032] The term "refractory" as used herein, refers to any subject
that does not respond with an expected clinical efficacy following
the delivery of a compound as normally observed by practicing
medical personnel.
[0033] The term "delivering" or "administering" as used herein,
refers to any route for providing a pharmaceutical or a
nutraceutical to a subject as accepted as standard by the medical
community. For example, the present invention contemplates routes
of delivering or administering that include, but are not limited
to, oral, transdermal, intravenous, intraperitoneal, intramuscular,
or subcutaneous.
[0034] The term "subject" as used herein, refers to any animal to
which an embodiment of the present invention may be delivered or
administered. For example, a subject may be a human, dog, cat, cow,
pig, horse, mouse, rat, gerbil, hamster etc.
[0035] The term "encapsulate", "encapsulated", or "encapsulating"
refers to any compound that is completely surrounded by a
protective material. For example, a compound may become
encapsulated by a population of nanoemulsion particle formation
during microfluidization.
[0036] The term "nutraceutical" refers to any compound added to a
dietary source (i.e., for example, a fortified food or a dietary
supplement) that provides health or medical benefits in addition to
its basic nutritional value.
[0037] The term "cosmeceutical" refers to any compound (i.e., for
example, benzoyl peroxide or retinol) added to a preparation that
possesses both cosmetic and pharmaceutical properties. A
cosmecuetical is generally useful for external applications to
improve the complexion or overall physical appearance.
Cosmeceuticals may be applied as compositions including, but not
limited to, a cream, oil, foam, spray, liquid etc. Cosmeceuticals
may include categories such as, but not limited to, carotenoids,
phenolic compounds, or water soluble antioxidants.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 presents exemplary data showing the particle diameter
distribution of a microfluidized plant sterol nanoemulsion
population three (3) months after preparation.
[0039] FIG. 1A presents exemplary data showing the particle
diameter distribution of a microfluidized plant sterol nanoemulsion
three (3) months after preparation.
[0040] FIG. 2 presents exemplary data showing the particle diameter
distribution of a microfluidized cod liver oil nanoemulsion
population four (4) months after preparation.
[0041] FIG. 3 presents exemplary data showing the particle diameter
distribution of a microfluidized tocopherol nanoemulsion population
five (5) months after preparation.
[0042] FIG. 4 presents exemplary data showing the particle diameter
distribution of a microfluidized lutein/zeaxanthin nanoemulsion
population.
[0043] FIG. 5 presents exemplary data showing the particle diameter
distribution of a microfluidized soy protein nanoemulsion
population.
[0044] FIG. 6 presents exemplary data showing the particle diameter
distribution of a microfluidized whey protein nanoemulsion
population.
[0045] FIG. 7 presents exemplary data showing the particle diameter
distribution of a microfluidized orange juice/plant sterol/lutein
nanoemulsion population.
[0046] FIG. 8 presents exemplary data showing the particle diameter
distribution of a microfluidized DHA fish oil/water nanoemulsion
population two (2) months after preparation.
[0047] FIG. 9 presents exemplary data showing the particle diameter
distribution of a microfluidized DHA fish oil/milk nanoemulsion
population three (3) weeks after preparation.
[0048] FIG. 10 presents exemplary data showing the particle
diameter distribution of a microfluidized DHA fish
oil/milk/tocopherol nanoemulsion population.
[0049] FIG. 11 presents exemplary data showing the particle
diameter distribution of a microfluidized DHA fish
oil/milk/tocopherol nanoemulsion population after
pasteurization.
[0050] FIG. 12 presents exemplary data showing the particle
diameter distribution of a microfluidized DHA fish
oil/milk/tocopherol nanoemulsion population after a freeze-thaw
process.
[0051] FIG. 13 presents exemplary data of gerbil plasma lycopene
levels when fed a lycopene-enriched diet.
[0052] FIG. 14 presents exemplary data of gerbil plasma lycopene
levels when fed a microfluidized lycopene nanoemulsion diet.
[0053] FIG. 15 presents one embodiment of an anti-bacterial
property generated during the preparation of a microfluidized plant
sterol nanoemulsion.
[0054] FIG. 16 presents exemplary data showing the particle
diameter distribution of a microfluidized plant sterol nanoemulsion
population used in Example 12.
[0055] FIG. 17 presents exemplary data showing that a
microfluidized plant sterol nanoemulsion diet is more effective in
reducing plasma LDL-C in hypercholesterolemic hamsters than either
a micronized plant sterol diet or a crystalline plant sterol diet
for four (4) weeks.
[0056] FIG. 18 presents exemplary data comparing premix cholesterol
particle diameter distributions from: Panel A: Tween.RTM. 80/Water
as per the '118 patent; and Panel B: Oil/Lecithin/Tween.RTM.
80/Water as contemplated by one embodiment of the present
invention.
[0057] FIG. 19 presents exemplary data comparing microfluidized
cholesterol nanoemulsion particle diameter distributions from:
Panel A: Tween.RTM. 80/Water as per the '118 patent using repeated
microfluidization passes; and Panel B: Oil/Lecithin/Tween.RTM.
80/Water as contemplated by one embodiment of the present invention
using a single microfluidization pass.
[0058] FIG. 20 presents exemplary data comparing microfluidized
cholesterol nanoemulsion particle diameter distributions from:
Panel A: Tween.RTM. 80/Water as per the '118 patent using a single
pass exposure; and Panel B: Oil/Lecithin/Tween.RTM. 80/Water as
contemplated by one embodiment of the present invention using a
single pass exposure.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention relates to the field of nanoemulsions.
In one embodiment, the nanoemulsion is created by a high shear
stress technology. In one embodiment, the invention comprises
uniform microfluidized nanoemulsions. In another embodiment, the
uniform nanoemulsion comprises a compound such as a pharmaceutical,
nutraceutical, or cosmeceutical. In one embodiment, the uniform
nanoemulsion comprises improved pharmacokinetic parameters when
compared to conventional nanoparticulate compositions and/or
nanoemulsions. In one embodiment, the present invention
contemplates a method of making a bacteria-resistant
nanoemulsion.
[0060] The use of nanoemulsions as a delivery system is generally
directed to pharmaceuticals. Nanoemulsion nutraceutical delivery,
however, has received little attention. For example, one
nanoemulsion system contains plant sterols. Bruce et al., "Method
for producing dispersible sterol and stanol compounds" U.S. Pat.
No. 6,387,411 (2002) (herein incorporated by reference). This
technology, however, uses a grinding method to produce the
nanoemulsions, and consequently, the particle diameter is at least
six (6) times greater than contemplated herein. Although it is not
necessary to understand the mechanism of an invention, it is
believed that this diameter difference offers particular advantages
in stability and efficacy (infra). Further, the '411 patent does
not disclose the incorporation of absorbable micronutrients.
[0061] A further use of nanoemulsions as a delivery system is
directed to cosmeceuticals. Cosmeceuticals may comprise, for
example; carotenoids including, but not limited to,
.alpha.-carotene, .beta.-carotene, .beta.-cryptoxanthin, lycopene,
crocetin, fucoxanthin, halocynthiaxanthin, canthaxanthin,
astraxanthin, lutein, or zeaxanthin; phenolic compounds including,
but not limited to, quercetin, rutin, myricetin, kaemferol,
catechin, epigallocatechin, epicatechin, reservatrol, tocopherol,
ferulate, ubiquinol-10, soy isoflavones such as genestein,
daidzein, alpha lipoic acid, anthocyanins, ellagic tannins, gallic
or ellagic acids; or water soluble anti-oxidants such as ascorbic
acid, uric acid, or bilirubin.
[0062] The present invention is directed to populations of
nanoparticles or nanoemulsions comprising an oral delivery vehicle
for all absorbable (i.e., for example, fat-soluble) nutrients
including, but not limited to, fatty acids, carotenoids,
tocopherols, tocotrienols, and coenzyme-Q. Delivery methods,
however, are not limited to oral and include, but are not limited
to, transdermal, intravenous, intraperitoneal, intramuscular, or
subcutaneous. In another embodiment, the carotenoids include, but
are not limited to, lutein and zeaxanthin. The present invention is
also directed to populations of nanoparticles or nanoemulsions
comprising an oral delivery vehicle for all non-absorbable (i.e.,
for example, fat soluble) plant sterol compounds including, but not
limited to, phytosterols and phytostanols. In one embodiment, the
compounds are encapsulated by the nanoparticles or nanoemulsions.
In one embodiment, common emulsifying agents are used to prepare
the nanoemulsions. In one embodiment, the emulsifying agents
include, but are not limited to, phospholipids, fatty acid
monoglycerides, fatty acid diglycerides, or polysorbates.
[0063] The present invention also contemplates that certain
nanoemulsion embodiments of the present invention comprise a
surface-to-volume ratio that results in an improved bioavailability
over current methods and compositions known in the art.
[0064] The present invention also contemplates that certain
nanoemulsion embodiments of the present invention are resistant to
microbiological growth. Although it is not necessary to understand
the mechanism of an invention, it is believed that the
microfluidization process comprises a high sheer stress and/or
creates an oxidizing environment, thereby disrupting microbial
integrity and/or preventing microbial growth.
1. Methods of Making Nanoemulsions
[0065] Nanoemulsions have been generated by a variety of methods.
In particular, these methods provide a wide variation in particle
diameter and require organic solvents and or polymers. When these
known nanoemulsions are considered for an oral drug or nutrient
delivery system, issues of biocompatibility and physiological side
effects become an important issue.
[0066] In one embodiment, the present invention contemplates a
method of making a nanoemulsion comprising a continuous turbulent
flow at high pressure. In one embodiment, the high pressure
turbulent flow comprises microfluidization. In one embodiment, a
uniform nanoemulsion is generated from a premix using a single pass
exposure (i.e., for example, within a thirty (30) second time
frame). In one embodiment, the uniform nanoemulsion comprises a
population of particles whose difference between the minimum and
maximum diameters does not exceed approximately 100 nm. In one
embodiment, a uniform nanoemulsion is generated using a pressure of
at least 25,000 PSI. In one embodiment, the present invention
contemplates a method of making uniform microfluidized
nanoemulsions without organic solvents or polymers. In one
embodiment, the microfluidized nanoemulsion is made from a
suspension. In another embodiment, the microfluidized nanoemulsion
is made from a microemulsion.
[0067] In one embodiment, the present invention contemplates a
uniform microfluidized nanoemulsion using compounds that are
substantially soluble in a liquid dispersion medium. In one
embodiment, the nanoemulsion encapsulated the compounds. In one
embodiment, the compounds comprise pharmaceuticals and/or
nutraceuticals.
[0068] Exemplary nutraceuticals and dietary supplements are
disclosed, for example, in Roberts et al., Nutriceuticals: The
Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing
Foods (American Nutriceutical Association, 2001), which is
specifically incorporated by reference. Dietary supplements and
nutraceuticals are also disclosed in Physicians' Desk Reference for
Nutritional Supplements, 1st Ed. (2001) and The Physicians' Desk
Reference for Herbal Medicines, 1st Ed. (2001), both of which are
also incorporated by reference. A nutraceutical or dietary
supplement, also known as a phytochemical or functional food, is
generally any one of a class of dietary supplements, vitamins,
minerals, herbs, or healing foods that have medical or biological
effects on the body.
[0069] Exemplary nutraceuticals or dietary supplements include, but
are not limited to, lutein, folic acid, fatty acids (e.g., DHA and
ARA), fruit and vegetable extracts, vitamin and mineral
supplements, phosphatidylserine, lipoic acid, melatonin,
glucosamine/chondroitin, Aloe Vera, Guggul, amino acids (e.g.,
glutamine, arginine, iso-leucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine), green tea,
lycopene, whole foods, food additives, herbs, phytonutrients,
antioxidants, flavonoid constituents of fruits, evening primrose
oil, flax seeds, fish and marine animal oils, and probiotics.
Nutraceuticals and dietary supplements also include bio-engineered
foods genetically engineered to have a desired property, also known
as "pharmafoods."
[0070] In particular, these compounds include, but are not limited
to, naturally occurring oils, fatty acids, and proteins. In one
embodiment, a naturally occurring oil comprises fish oil (i.e., for
example, cod liver oil). In one embodiment, a naturally occurring
fatty acid comprises an omega-3 (i.e., for example, DHA). In one
embodiment, the nanoemulsion comprises little or no fat. In one
embodiment, a naturally occurring protein comprises soy or
whey.
[0071] In one embodiment, the present invention contemplates a
method of making a uniform microfluidized nanoemulsion comprising a
population of particles whose diameter ranges from between 10-110
nm.
[0072] A. The Microfluidizer
[0073] Microfluidization is a unique process that powers a single
acting intensifier pump. The intensifier pump amplifies the
hydraulic pressure to the selected level which, in turn, imparts
that pressure to the product stream. As the pump travels through
its pressure stroke, it drives the product at constant pressure
through the interaction chamber. Within the interaction chamber are
specially designed fixed-geometry microchannels through which the
product stream will accelerate to high velocities, creating high
shear and impact forces that generates a uniform nanoemulsion as
the high velocity product stream impinges on itself and on
wear-resistant surfaces.
[0074] As the intensifier pump completes its pressure stroke, it
reverses direction and draws in a new volume of product. At the end
of the intake stroke, it again reverses direction and drives the
product at constant pressures, thereby repeating the process.
[0075] Upon exiting the interaction chamber, the product flows
through an onboard heat exchanger which regulates the product to a
desired temperature. At this point, the product may be recirculated
through the system for further processing or directed externally to
the next step in the process. Cook et al., "Apparatus For Forming
Emulsions" U.S. Pat. No. 4,533,254 (1985); and Cook et al., "Method
Of Forming A Microemulsion" U.S. Pat. No. 4,908,154 (1990) (both
herein incorporated by reference).
[0076] B. Nanoparticulate Compositions
[0077] Early attempts using microfluidizers to create
nanoparticulate compositions required drug substances that were
poorly soluble in a liquid dispersion medium. In one disclosed
technology, "poorly soluble" was defined as less than 10 mg/ml.
Bosch et al., "Process for preparing therapeutic compositions
containing nanoparticles" U.S. Pat. No. 5,510,118 (1996) (herein
incorporated by reference). While water-insolubility was preferably
considered, oil-insoluble compounds were also subjected to a
microfluidization process. The '118 patent's microfluidization
process is described as a "milling" action, thus indicating that
the insoluble compound particles are undergoing a physical
disintegration during the creation of the nanoparticulate
composition. Further, this previous process requires
long-processing times (i.e., repeated microemulsifying cycles)
thereby promoting heat build-up in the microfluidizer.
Consequently, this early technique requires processing temperatures
of less than 40.degree. C. One problem is that this technique
resulted in average nanoemulsion particle diameters of
approximately 300 nm. Despite teachings within the '118 patent that
lower particle diameters (i.e., less than 100 nm) can be achieved,
no data is presented demonstrating such a capability. Exemplary
data presented herein has used the Bosch et al. process to produce
a complete particle diameter distribution profile. See Example 13.
These data show that the Bosch et al. technology cannot produce a
uniform nanoemulsion as contemplated by the present invention.
[0078] Several others have implemented the basic '118 technology to
encapsulate various insoluble compounds. In fact, these subsequent
disclosures define a nanoparticle composition as "particles
consisting of a poorly soluble therapeutic or diagnostic agent
having adsorbed onto, or associated with, the surface thereof a
non-crosslinked surface stabilizer". Cooper et al.,
"Nanoparticulate Sterol Formulations And Novel Sterol Combinations"
United States Patent Application Publication No. 2004/0033202 A1
(2004) (see pg 1 para 3) (herein incorporated by reference). Like
the '118 patent, Cooper et al. discloses preparing nanoparticulate
compositions using compounds that are poorly soluble in a liquid
dispersion medium (i.e., water, oils, alcohols, glycols, etc.). For
example, Cooper et al. defines a "poorly water-soluble" drug as
having a solubility of less than about 30 mg/ml. For example, plant
sterol nanoparticulate compositions comprising one or more sterols
or stanols (i.e., sitosterol or phytosterols) are suggested in the
art as having a particle diameter of less than 50 nm. Cooper et al.
does not use a microfluidizer nor present any data showing a
capability of providing a uniform particle diameter ranging between
10-110 nm. Instead, Cooper et al. relies upon a more traditional
milling process that does not produce a uniform particle diameter
distribution ranging between 10-110 nm.
[0079] Cooper et al. employs a milling grinder known in the art as
a DYNO.RTM.-MILL KDL. This equipment is currently marketed in the
United States by Glen Mills, Inc. (Clifton, N.J.) and advertises
with the following technical information. The DYNO.RTM.-MILL is a
versatile horizontal bead mill having applications ranging from
paints and coatings to drug manufacturing and cell disruption for
extracting proteins. Grinding to a mean diameter of 320 nm has been
reported in research papers. Operation of the DYNO.RTM.-MILL is
always wet, that is, the material to be ground is held in
suspension in any suitable liquid. A jacketed grinding chamber
contains a series of agitators that are equally spaced along the
length of a central shaft. The jacket on the grinding chamber is
used to control the temperature of the material being processed.
The chamber is secured at one end and cantilevers out over the
shaft. The bearing end contains a separator gap which has clearance
tolerances that can be set as tight as 20 microns. The chamber is
filled to about 80% of its capacity with beads (i.e., PolyMill.RTM.
500; 500 .mu.m diameter grinding beads). Depending on the specific
application beads made from glass, ceramic, metals, tungsten
carbide and other materials are available. The process material is
now introduced into the chamber. When the chamber is full of
material and beads, the machine is switched on and the agitator
discs rotate forcing the beads to impact over and over with the
process material with hurricane-like force. This action of having
thousands of separate impacts produces rapid and consistent size
reduction. Batch and continuous processing can be handled in the
same mill by changing the grinding chamber and the gap setting.
[0080] At best, Cooper et al. is limited to a plant sterol
nanoparticulate composition where 90% of the particle diameters are
below 187 nm. The actual particle diameter distribution, however,
is not presented. In one embodiment, the present invention
contemplates that the technology described by Cooper et al. cannot
produce a uniform particle diameter distribution ranging between
10-110 nm. See Example 14. Unlike some embodiments of the present
invention, Cooper et al. has not considered methods to make a
nanoparticulate composition that include a heating process. In
fact, Cooper et al. presents a discussion concluding that preparing
a plant sterol nanoparticulate composition using a process that
includes heating is not desirable and problematic. Some embodiments
of the present invention have solved those problems.
[0081] Two drugs that are insoluble in a selected liquid dispersion
medium, meloxicam and topiramate, are suggested as potential
candidates for improved clinical administration using the Cooper et
al. nanoparticulate composition technology. Cooper et al.,
"Nanoparticulate meloxicam formulations" US Pat. Appln Publ. No.
2004/0229038 (2004); and Gustow et al., "Nanoparticulate topiramate
formulations" US Pat. Appln Publ. No. 2004/0258758 (2004). Neither
publication contains any exemplary data demonstrating the creation
of a uniform microfluidized microemulsion having a particle
diameter range of about 10-110 nm.
[0082] C. Nanoemulsification
[0083] The formation of a uniform mixture (i.e., for example, a
population) of predominantly small particles may involve a physical
process termed "emulsification". An emulsion is traditionally
defined in the art "as a system . . . consisting of a liquid
dispersed with or without an emulsifier in an immiscible liquid
usually in droplets of larger than colloidal size" Medline Plus
Online Medical Dictionary, Merriam Webster (2005). Consequently, as
the art developed emulsifiers capable of generating smaller and
smaller diameter particles, the terms "microemulsion" and
"nanoemulsion" became known. Conceptually, a microemulsion is one
thousand-fold greater in diameter than a nanoemulsion. However,
particle diameter distributions may vary widely in a non-controlled
emulsification process creating considerable overlap between the
nanoemulsion and microemulsion technologies.
[0084] In one embodiment, the present invention contemplates a
premix comprising a compound substantially soluble (i.e., for
example, greater than 30 mg/ml) in a liquid dispersion medium
(i.e., for example, a heated liquid dispersion medium) and,
optionally, common emulsifying agents including, but not limited
to, phospholipids, fatty acid monoglycerides, fatty acid
diglycerides, or polysorbates. In one embodiment, a nanoemulsion is
created by exposing a premix to a continuous turbulent flow at a
high pressure, wherein the pressure is at least 25,000 PSI. In one
embodiment, the high pressure turbulent flow comprises
microfluidization. In one embodiment, the nanoemulsion comprises
particles encapsulating pharmaceuticals or nutraceuticals. In one
embodiment, the nanoemulsion comprises a uniform nanoemulsion
having stable particles. In one embodiment, the microfluidization
comprises a single pass exposure (i.e., for example, approximately
thirty (30) seconds). In one embodiment, a uniform plant sterol
microfluidized nanoemulsion has an improved low density lipoprotein
cholesterol lowering efficacy.
[0085] Oral drug administration is a common method for providing
pharmaceuticals and nutraceuticals to any subject. The contemplated
methods of delivering a nanoemulsion is not limited to oral and
include, for example, transdermal, intravenous, intraperitoneal,
intramuscular, or subcutaneous routes of administration. Oral
administration is favored because the formulations (i.e., liquids
or suspensions) are relatively inexpensive to produce and are well
tolerated. Subsequent gastrointestinal absorption of the
formulation's ingredients, however, is not as predictable. For the
pharmaceuticals and nutraccuticals to gain entrance into the
subject, the formulations must be compatible with the digestive
system. Consequently, lipid-based drug delivery systems are known
to be useful as carriers for many drug delivery systems. Their
efficacy, however, may be dependent upon; i) lipid composition
(i.e., for example, molecular size and charge); ii) pharmaceutical,
nutraceutical, or cosmeceutical chemical structure (i.e., molecular
size and pH ionization); and iii) the overall health of the
subject. Lipids are generally categorized as physiologically
non-absorbable or absorbable. It should be recognized that
gastrointestinal absorption processes are unrelated to a compound's
solubility properties. The present invention contemplates
compositions and methods related to uniform microfluidized
nanoemulsions comprising either absorbable or non-absorbable lipids
thereby improving their bioavailability.
[0086] 1. Non-Absorbable Lipids
[0087] Plant sterols, stanols, and triterpene alcohols (i.e., for
example, oryzanol) are either not absorbed, or poorly absorbed,
into the bloodstream following oral administration. In one
embodiment, the present invention contemplates a method of making a
uniform nanoemulsion (i.e., for example, microfluidized) comprising
a non-absorbable lipid having substantial solubility in a liquid
dispersion medium and, optionally, common emulsifying agents, such
as phospholipids, fatty acid monoglycerides, fatty acid
diglycerides, or polysorbates to formulate improved nanoemulsions.
In one embodiment, the nanoemulsion comprises particle diameters
ranging between 10-110 nm, thereby improving oral
administration.
[0088] The use of plant sterols, such as .beta.-sitosterol, is
known to reduce blood cholesterol levels because it is
non-absorbable. The presence of unabsorbed plant sterols in the
gastrointestinal system inhibits the normal metabolism of
cholesterol and, concomitantly, decreases blood cholesterol levels.
Specifically, administration of twenty (20) gms of crystalline
plant sterols can reduce plasma cholesterol levels approximately
10%. Pollack et al., "Sitosterol", In: Monographs on
Atherosclerosis, Vol. 10, Eds. O. J. Pollack & D. Kritchevsky,
Basel, N.Y., Karger (1981).
[0089] Further, non-absorbable lipids are advantageous as a
nutraceutical because of a lack of side effects. Side effects are
routinely observed when using traditional pharmaceutical systemic
cholesterol-lowering interventions (i.e., for example, HMG CoA
reductase inhibitors or niacin). Because of the very low incidence
of side effects, plant sterols can be prescribed for the general
population, including children for whom systemic interventions are
rarely recommended. It is known that the consumption of adequate
amounts of plant sterols will lower blood cholesterol levels. The
present invention contemplates improvements in currently known
methods to deliver plant sterols or stanols.
[0090] The first known method involves dissolving the plant sterol
in a vegetable oil-containing margarine to an efficacious level of
plant sterol. When the fat solubility of a free stanol or a sterol
is increased by: i) interesterified with a fatty acid such oleate
or linoleate; ii) mixed in vegetable oil; or iii) hydrogenated to
produce margarine, plasma cholesterol can be reduced by
approximately 30%. To ingest enough plant sterol, this process can
result in the consumption of up to approximately eighteen (18)
grams of fat. Miettinen et al., "Use of a stanol fatty acid ester
for reducing serum cholesterol level" U.S. Pat. No. 5,502,045
(1996); and Wester et al., "Phytosterol compositions" U.S. Pat. No.
6,589,588 (2003) (both herein incorporated by reference). To fat
conscious Americans, coupled with the high cost of the margarines,
this is unacceptable for a naturopathic approach to lower plasma
cholesterol. A disadvantage of this method is that overweight or
obese people frequently have elevated cholesterol levels.
Physicians, of course, caution this subject group to avoid
additional dietary fat. In one embodiment, the present invention
contemplates a method of making a beverage nanoemulsion that
comprises plant sterols. For example, the method to make the
beverage nanoemulsion may comprise a continuous turbulent flow at a
high pressure. In one embodiment, the continuous turbulent high
pressure flow comprises microfluidization. In another embodiment,
the nanoemulsion beverage comprises an orange juice product.
[0091] The second known method comprises oral delivery of
water-dispersible plant sterols (i.e., for example, a stanol not
dissolved in fat) by incorporation micron-sized micelles (i.e.,
microemulsions having diameters of several thousand nanometers)
which can be subsequently added to beverages or foods. Ostlund,
Jr., "Sitostanol formulation to reduce cholesterol absorption and
method for preparing and use of same" U.S. Pat. No. 5,932,562
(1999)(herein incorporated by reference). When the microemulsion
containing the plant sterol was administered into the intestine,
cholesterol absorption was reduced by approximately 37%. Ostlund,
Jr., "Sitostanol formulation to reduce cholesterol absorption and
method for preparing and use of same" U.S. Pat. No. 5,932,562
(1999)(herein incorporated by reference); and Spillburg et al.,
"Fat-free foods supplemented with soy stanol-lecithin powder reduce
cholesterol absorption and LDL cholesterol" J Am Diet Assoc.
103:577-581 (2003). A disadvantage of this method is that the
particle diameters of these microemulsion preparations are on the
order of thousands of nanometers (i.e., micron diameters) and
thereby does not provide optimal efficacy. The present invention
contemplates a nanoemulsion technology comprising a specific
formulation and a microfluidization process that provides particle
diameters from between 10-110 nm. In one embodiment, the
nanoparticle has improved pH and temperature stability properties,
thereby stabilizing the particle's integrity throughout the
gastrointestinal system.
[0092] The third known method involves the oral delivery of plant
sterols by producing a water dispersible sterol product. These
water dispersible products usually include emulsifying agents
including, but not limited to, monoglycerides and polysorbates.
These water dispersible products are known to be homogenized using
a liquid/liquid dispersion having particle diameters less than 1000
nm (mean=358 nm). The present invention, however, contemplates a
microfluidizing nanoemulsion technology (i.e., for example, that
produced by a continuous flow high pressure process) that improves
the emulsification of these water-dispersible plant sterols into
nanoemulsions having a particle diameter of approximately 40-60
nm.
[0093] Similarly, methods are known for preparing water dispersible
sterol/stanol or sterol/stanol ester compositions by co-melting the
stanol/sterols with highly branched hydrocarbons and then grinding
the resulting product. Bruce et al., "Method for producing
dispersible sterol and stanol compounds" U.S. Pat. No. 6,387,411
(2002) (herein incorporated by reference). This grinding method
typically produces particle diameters ranging from 10-150 microns.
Other methods known to produce a water dispersible sterol product
use homogenization in emulsifying agents including, but not limited
to, monoglycerides and polysorbates. These homogenization
procedures have been reported to produce a liquid/liquid dispersion
with a particle diameter less than 1000 nm (mean=358 nm). Stevens
et al., "Aqueous dispersible sterol product" U.S. Pat. No.
6,623,780 (2003) (herein incorporated by reference). This
preparation, when added to orange juice, can reduce LDL cholesterol
by approximately 12%. Devaraj et al., "Plant sterol-fortified
orange juice effectively lowers cholesterol levels in mildly
hypercholesterolemic healthy individuals" Arterioscler Thromb Vasc
Biol. 24:25-28 (2004).
[0094] Although it is not necessary to understand the mechanism of
an invention, it is believed that a much greater surface-to-volume
ratio is reached in the uniform microfluidized nanoemulsion
preparations made according to the present invention (i.e., for
example, up to 6 fold) and results in greater stability.
Consequently, it is further believed that, any incorporated
pharmaceutical, nutraceutical, or cosmeceutical has improved
efficacy (i.e., for example, plasma cholesterol lowering by a plant
sterol). It is further believed that a smaller-sized plant
sterol-containing nanoparticles contemplated by one embodiment of
the present invention, when compared to known micron-sized micelles
or microemulsions, has an improved disruption of the normal
micellar delivery of dietary cholesterol to the digestive tract.
For example, it is known that pre-formed micron-size micelles
containing plant stanols were up to three (3) times more
efficacious in inhibiting cholesterol absorption than a suspension
of crystalline stanol. Ostlund et al., "Sitostanol administered in
lecithin micelles potently reduces cholesterol absorption in
humans" Am J Clin Nutr 70:826-831 (1999).
[0095] 2. Absorbable Lipids
[0096] This invention also relates to the use of nanoemulsions as
an oral delivery vehicle for absorbable lipids including, but not
limited to, fatty acids, carotenoids, tocopherols and other fat
soluble vitamins, tocotrienols, and Coenzyme-Q. In one embodiment,
the present invention contemplates a method to make a uniform
microfluidized nanoemulsion comprising an absorbable lipid having
substantial solubility in a liquid dispersion medium and,
optionally, common emulsifying agents, such as phospholipids, fatty
acid monoglycerides, fatty acid diglycerides, or polysorbates to
formulate improved nanoemulsions. In one embodiment, the method
comprises a step exposing a premix to a continuous turbulent flow
at high pressure. In one embodiment, the pressure is at least
25,000 PSI. In one embodiment, the nanoemulsion comprises
carotenoids, including, but not limited to, lutein and zeaxanthin.
In one embodiment, the nanoemulsion comprises nanoparticles having
a particle diameter ranging from 10-110 nm, thereby improving
bioavailability. In one embodiment, nanoemulsion bioavailability is
improved following oral, transdermal, intravenous, intraperitoneal,
intramuscular or subcutaneous delivery.
[0097] In one embodiment, the present invention contemplates a
method to treat or prevent macular degeneration (i.e., a major
cause of blindness in people of 65) providing an improved
nanoemulsion comprising at least one carotenoid. In one embodiment,
the carotenoid is selected from the group comprising lutein or
zeaxanthin.
[0098] Under normal physiological conditions these types of
compounds may be poorly absorbed by the gastrointestinal system.
Consequently predicable lipid nutrient absorption is highly
variable thus resulting in a highly variable lipid bioavailability
(i.e., for example, the percentage of the dose absorbed). Factors
influencing bioavailability may include, but are not limited to,
food processing methods, food matrix, and physiological solubility
in naturally-occurring micelles (i.e., for example, the lipid
micellular transport system).
[0099] Fat-soluble nutrients can be incorporated into high
fat-containing vegetable oils for dispersal into a fat matrix
(i.e., for example a micron-sized micelle). The micelle solubilizes
the lipid-soluble nutrient thereby allowing absorption by the small
intestine. For example, when plant sterols are delivered in a
micelle, cholesterol absorption inhibition is increased up to
three-fold. Ostlund et al., "Sitostanol administered in lecithin
micelles potently reduces cholesterol absorption in humans" Am J
Clin Nutr 70:826-831 (1999).
[0100] Similarly, an increased in vitro carotenoid bioavailability
in cell cultures is observed when solubilizing the carotenoids in
micelles. Xu et al., "Solubilization and stabilization of
carotenoids using micelles: delivery of lycopene to cells in
culture" Lipids 34:1031-1036 (1999). A disadvantage of using
micelles, however, involves the use of chlorinated organic
solvents, a practice that should be avoided in the processing of
foods stuffs. Another in vitro experiment demonstrates that a
nanoemulsion preparation of lipophilic substances, such as fatty
acids, vitamins, and beta-carotene can be delivered into cell
culture medium (RPMI-1640) and incorporated by TK-6 cells. Zuelli
et al., "Delivering lipophilic substances into cells using
nanoemulsions" U.S. Pat. No. 6,558,941 (2003) (herein incorporated
by reference).
II. Uniform Nanoemulsion Pharmacokinetics
[0101] In one embodiment, the present invention contemplates a
nanoemulsion produced by a continuous turbulent flow at high
pressure having improved pharmacokinetic properties when compared
to conventional nanoparticulate compositions and/or nanoemulsions
currently known in the art. It is known that nanoparticles deliver
and/or release drugs (i.e., for example, norflaxin) and/or proteins
(i.e., for example, serum albumin) more effectively than
microparticles. Jeon et al., "Effect of solvent on the preparation
of surfactant-free poly(DL-lactide-co-glycolide) nanoparticles and
norfloxacin release characteristics` Int J Pharm 207; 99-108
(2000); and Panyam et al., "Polymer degradation and in vitro
release of a model protein from poly(D,L-lactide-co-glycolide)
nano- and microparticles" J Control Release 92:173-187 (2003).
[0102] One embodiment of the present invention contemplates a
uniform microfluidized nanoemulsion having improved pharmacokinetic
properties when compared to conventional nanoparticulate
compositions and/or nanoemulsions currently known in the art. One
advantage of uniform microfluidized nanoemulsions comprises a
narrow particle diameter range (i.e., for example, 10-110 nm). Most
conventional nanoparticle compositions and/or nanoemulsions
currently known have a wide distribution of particle diameters that
interfere with the improved efficacies and bioavailabilities of the
smaller sized particles.
[0103] The present invention has solved the problem of generating
nanoemulsions with highly variable particle diameters and provides
a more uniformly small-sized nanoemulsions (i.e., for example, a
uniform nanoemulsion comprising stable particles). Consequently,
these uniform nanoemulsions provide improved pharmacokinetic
parameters when compared to conventional nanoparticle compositions
and/or nanoemulsions currently known in the art independent of the
mode of delivery which includes, but is not limited to, oral,
transdermal, intravenous, intraperitoneal, intramuscular,
subcutaneous, etc.
[0104] A. Absorption Phase
[0105] The use of conventional nanoparticulate compositions or
nanoemulsions is not ideal due to delayed onset of action. In
contrast, a uniform microfluidized nanoemulsion as contemplated by
the present invention exhibits faster therapeutic effects.
[0106] Pharmaceuticals and nutraceuticals are commercially
available as tablets, liquids, gel caps, capsules etc., generally
intended for oral administration. Peak plasma concentrations of
these compositions usually occur between 2-4 hours following
administration.
[0107] When a uniform microfluidized nanoemulsion contemplated by
the present invention is formulated into an oral dosage form peak
plasma concentrations of an incorporated compound can be obtained
in less than about 2 hours, preferably less than about 1 hour, more
preferably less than about 30 minutes, but most preferably between
1 and 15 minutes.
[0108] B. Frequency of Dosing and Dosage Quantity
[0109] The recommended total daily dose of most pharmaceuticals and
nutraceuticals are administered in divided doses. It is known in
the art that a single daily dose may be preferable to multiple dose
each day. For example, in studies of adults with partial onset
seizures, a daily dose of 200 mg/day has inconsistent effects and
is less effective than 400 mg/day. See Physicians' Desk Reference,
57.sup.th Edition, pp. 2502 (2003).
[0110] In contrast, some uniform microfluidized nanoemulsions of
the present invention may be administered less frequently, at lower
doses, and in dosage forms such as liquid dispersions, powders,
sprays, solid re-dispersible dosage forms, ointments, creams, etc.
Exemplary types of formulations useful in the present invention
include, but are not limited to, liquid dispersions, gels, aerosols
(pulmonary and nasal), ointments, creams, solid dose forms, etc. of
any pharmaceutical, nutraceutical, or cosmeceutical. Lower dosages
can be used because the smaller particle diameters of embodiments
of the present invention ensure more complete absorption.
[0111] In one embodiment, the present invention contemplates a
therapeutically effective amount of a uniform microfluidized
nanoemulsion having 1/6, 1/5, 1/4, 1/3, or 1/2 of the
therapeutically effective amount of a conventional pharmaceutical,
nutraceutical, or cosmeceutical formulations.
[0112] C. Oral Administration
[0113] A liquid dosage form of a conventional nanoparticulate or
nanoemulsion composition would be expected to be a relatively large
volume, highly viscous substance which would not be well accepted
by subject populations. Moreover, viscous solutions can be
problematic in parenteral administration because these solutions
require a slow syringe push and can stick to tubing. In addition,
conventional formulations of poorly water-soluble active agents
tend to be unsafe for intravenous administration techniques, which
are used primarily in conjunction with highly water-soluble
substances. Embodiment contemplated by the present invention solves
this problem by utilizing a liquid dispersion medium in which the
pharmaceutical, nutraceutical, or cosmeceutical is substantially
soluble.
[0114] Liquid dosage forms of embodiments of a uniform
microfluidized nanoemulsion provide significant advantages over a
liquid dosage form of a conventional nanoparticulate or
nanoemulsion. In one embodiment, the uniform microfluidized
nanoemulsion comprises a low viscosity. In another embodiment, the
uniform nanoemulsion comprises a silky texture. These advantages
include, for example: i) better subject compliance due to the
perception of a lighter formulation which is easier to consume and
digest; ii) ease of dispensing because one can use a cup or a
syringe; iii) potential for formulating a higher concentration of a
pharmaceutical, nutraceutical, or cosmeceutical resulting in a
smaller dosage volume and thus less volume for the subject to
consume; and iv) easier overall formulation concerns.
[0115] Liquid formulations of uniform nanoemulsions contemplated by
the present invention are easier to consume which is especially
important when considering juvenile subjects, terminally ill
subjects, and elderly subjects. Viscous or gritty formulations, and
those that require a relatively large dosage volume, are not well
tolerated by these subject populations. Liquid oral dosage forms
can be particularly preferably for subject populations who have
difficulty consuming tablets, such as infants and the elderly.
[0116] The viscosities of liquid dosage forms of nanoparticulate
topiramate according to the invention are preferably less than
about 1/200, less than about 1/175, less than about 1/150, less
than about 1/125, less than about 1/100, less than about 1/75, less
than about fraction 1/50, or less than about 1/25 of a liquid oral
dosage form of a conventional nanoparticulate composition or
nanoemulsion at about the same concentration per ml.
[0117] In one embodiment, the present invention contemplates a
uniform microfluidized nanoemulsion that is not turbid. In one
embodiment, turbid refers to the property of particulate matter
that can be seen with the naked eye or that which can be felt as
"gritty" when consumed. Embodiments of nanoemulsions contemplated
by the present invention can be poured out of or extracted from a
container as easily as water, whereas a liquid dosage form of a
conventional nanoparticulate or nanoemulsion composition is
expected to exhibit notably more "sluggish" characteristics.
[0118] D. Increased Bioavailability
[0119] In one embodiment, the present invention contemplates a
uniform microfluidized nanoemulsion having an increased
bioavailability and a smaller dose requirement as compared to prior
conventional nanoparticulate compositions and nanoemulsions
administered at the same dose.
[0120] Any pharmaceutical, nutraceutical, or cosmeceutical can have
adverse side effects if administered at a specific dose for a
specific duration. Thus, lower doses which can achieve the same or
better therapeutic effects as those observed with larger doses are
desired. Such lower doses may be realized with a uniform
microfluidized nanoemulsion contemplated by the present invention
due to greater bioavailability as compared to conventional
nanoparticulate compositions and nanoemulsions; consequently
smaller dose of pharmaceuticals and nutraceutical are likely
required to obtain the desired therapeutic effect.
[0121] For example, the relative bioavailability of pharmaceutical,
nutraceutical, or cosmeceutical incorporated into a conventional
nanoparticulate or nanoemulsion may be about 85% (i.e., as compared
to a pure solution). In one embodiment, a uniform microfluidized
nanoemulsion formulated into an oral pharmaceutical, nutraceutical,
or cosmeceutical dosage form has a relative bioavailability
preferably greater than about 85%. In other embodiments, the
relative bioavailability is greater than about 90%, or greater than
about 95%, or greater than about 98%.
[0122] E. Pharmacokinetic Profiles
[0123] The present invention also provides embodiments of uniform
microfluidized nanoemulsions having incorporated pharmaceuticals
and/or nutraceuticals having improved pharmacokinetic profiles when
administered to mammalian subject. In one embodiment, the improved
profile is compared to conventional nanoparticulate compositions
and nanoemulsions.
[0124] An improved pharmacokinetic (pK) profile according to the
present invention can have several different types of attributes.
In one embodiment, an improved pK profile of a uniform
microfluidized nanoemulsion may produce the same pK profile as a
conventional nanoparticulate composition or nanoemulsion, but at a
lower dose. In another embodiment, an improved pK profile requires
less frequent dosing as compared to a conventional nanoparticulate
composition or nanoemulsion. In one embodiment, an improved pK
profile shows a faster onset of activity and/or greater quantity of
drug absorbed (i.e., greater bioavailability) than conventional
nanoparticulate compositions and nanoemulsions. In another
embodiment, an improved pK profile allows a more effective and/or
faster titration of the subject to therapeutic plasma levels.
[0125] The present invention contemplates certain embodiments of
uniform microfluidized nanoemulsions comprising an improved
pharmacokinetic profile as reflected by
time-to-maximum-concentration (T.sub.max), maximum-concentration
(C.sub.max), and/or area-under-curve (AUC) profiles.
[0126] In one embodiment, an administered dose of a pharmaceutical,
nutraceutical, or cosmeceutical incorporated into a uniform
microfluidized nanoemulsion comprises a T.sub.max less than that of
a conventional nanoparticulate composition and/or nanoemulsion,
administered at the same dosage. Preferably the T.sub.max is less
than about 99%, less than about 90%, less than about 80%, less than
about 70%, less than about 60%, less than about 50%, less than
about 40%, less than about 30%, less than about 25%, less than
about 20%, less than about 15%, or less than about 10% of the
T.sub.max of a conventional nanoparticulate composition and/or
nanoemulsion, administered at the same dosage.
[0127] In another embodiment, an administered dose of a
pharmaceutical, nutraceutical, or cosmeceutical incorporated into a
uniform microfluidized nanoemulsion comprises a C.sub.max greater
than that of a conventional nanoparticulate composition and/or
nanoemulsion, administered at the same dosage. Preferably, the
C.sub.max is greater than about 5%, greater than about 10%, greater
than about 15%, greater than about 20%, greater than about 30%,
greater than about 40%, greater than about 50%, greater than about
60%, greater than about 70%, greater than about 80%, greater than
about 90%, greater than about 100%, greater than about 110%,
greater than about 120%, greater than about 130%, greater than
about 140%, or greater than about 150% than the C.sub.max of a
conventional nanoparticulate composition and/or nanoemulsion,
administered at the same dosage.
[0128] In one embodiment, an administered dose of a pharmaceutical,
nutraceutical, or cosmeceutical incorporated into a uniform
microfluidized nanoemulsion comprises an AUC greater than that of a
conventional nanoparticulate composition and/or nanoemulsion,
administered at the same dosage. Preferably, the AUC is greater
than about 5%, greater than about 10%, greater than about 15%,
greater than about 20%, greater than about 30%, greater than about
40%, greater than about 50%, greater than about 60%, greater than
about 70%, greater than about 80%, greater than about 90%, greater
than about 100%, greater than about 110%, greater than about 120%,
greater than about 130%, greater than about 140%, or greater than
about 150% than the AUC of a conventional nanoparticulate
composition and/or nanoemulsion, administered at the same
dosage.
III. Sterile Nanoemulsions
[0129] The present invention contemplates a method of making a
nanoemulsion having anti-bacterial properties. In one embodiment,
the method comprises a step exposing a premix to a continuous
turbulent flow at high pressure. In one embodiment, the
anti-bacterial nanoemulsion is prepared by microfluidization. In
one embodiment, the exposing comprises approximately thirty (30)
seconds. In another embodiment, the exposing comprises a pressure
of at least 25,000 PSI. In another embodiment, the anti-bacterial
nanoemulsion comprises soy protein.
[0130] For example, a powdered soy protein preparation was added to
water thus creating a suspension. Then, a first aliquot of the
suspension was added to a first container (i.e., for example, a
cell culture falcon flask) that served as a control. A second
aliquot of the suspension was microfluidized (supra) to create a
nanoemulsion. The preparation was microfluidized in accordance with
Example 5. The microfluidized nanoemulsion was then added to a
second container. Both containers were refrigerated immediately and
observed over the next several days. The control suspensions
agglomerated and grew bacteria. See FIGS. 15A and 15B. In contrast,
the microfluidized nanoemulsion containing the soy protein did not
agglomerate or grow bacteria. See FIGS. 15C and 15D.
[0131] Although it is not necessary to understand the mechanism of
an invention, it is believed that the microfluidization sterilized
the bacteria. It is further believed that the microfluidization
shear stress resulted in a bacterial cell lysis thereby preventing
further bacterial growth. Consequently, it is believed that
microfluidization, as contemplated herein, produces a
microbiologically sterile composition.
[0132] In one embodiment, the present invention contemplates a
nanoemulsion comprising an oxidizing environment produced by a
method comprising a continuous turbulent flow at a high pressure.
In one embodiment, the nanoemulsion comprises a uniform
microfluidized nanoemulsion. In one embodiment, the oxidizing
environment prevents bacterial growth. In another embodiment, the
oxidizing environment is bacteriocidal. In another embodiment, the
oxidizing environment provides a sterile nanoemulsion.
[0133] An oxidizing nanoemulsion environment may result from an
increased surface to volume ratio. In one embodiment, the present
invention also contemplates a method to avoid the generation of an
oxidizing environment by microfluidizing in the presence of an
antioxidant. In one embodiment, the antioxidant reduces the
presence of reactive oxygen species (ROS) in the microfluidized
nanoemulsion. In another embodiment, the
TABLE-US-00001 Sample Sample Sample Formulation 1 2 3 Mean Plasma
4.0 2.8 3.3 3.4 (unoxidized control) Plasma + FeCl.sub.3 12.4 16.0
13.1 13.9 (oxidized control) 1.5 g DHA with 200 ml milk 44.0 42.6
45.8 44.1 (microfluidized) 1.75 g DHA, 1000 mg Vit E 12.8 19.2 20.1
17.4 and 800 mg Vit C with 200 ml milk (not microfluidized) 1.75 g
DHA, 1000 mg Vit E 4.0 6.0 3.5 4.5 and 800 mg Vit C with 200 ml
milk (microfluidized) 1.75 g DHA and 800 mg Vit C 17.5 17.8 20.8
18.7 with 200 ml milk (microfluidized) 1.75 g DHA and 1000 mg Vit E
9.8 16.7 11.4 12.6 with 200 ml milk (microfluidized)
antioxidants are encapsulated by the nanoparticles for subsequent
release to the subject.
[0134] The ROS load within any nanoemulsion preparation can be
quantitatively determined by measuring indicators of an oxidizing
environment. Malondialdehyde (MDA), is a known indicator of an
oxidizing environment.
Table 1: Oxidative Stress in Nanoemulsion Formulations as Measured
by Malondialdehyde Formation
[0135] As can be seen in Table 1 above, the process of making a
microfluidized nanoemulsion increases MDA levels by approximately
13-fold. Further, the presence of both vitamin C and/or vitamin E
completely prevented MDA generation in microfluidized nanoemulsions
thereby returning MDA to homeostatic plasma levels.
EXPERIMENTAL
[0136] The following examples are specific embodiments as
contemplated by the present invention and are not intended to be
limiting.
Example 1
Stable Formulation of Plant Sterol Microfluidized Nanoemulsions
[0137] This example presents one plant sterol embodiment of a
microfluidized nanoemulsion. The step-wise procedure is as
follows:
[0138] 1. Heat 4 g of soybean oil
[0139] 2. Add 5 g soy lecithin, stir and heat to 90.degree. C.
[0140] 3. Add 1 g plant sterol, stir and heat 10 mins
[0141] 4. Add 250 mg polysorbate 80.
[0142] 5. Heat 240 mL de-ionized water to 70.degree. C.
[0143] 6. Add step 4 mixture to step 5 mixture, keep stir bar and
heat on for 30 mins
[0144] 7. Homogenize step 6 mixture for 2-4 mins
[0145] 8. Stir formulation for 10 mins on hot plate
[0146] 9. Microfluidize using a M-110EH unit once at 25,000 PSI
[0147] 10. Do particle diameter analysis using a Malvern Nano S
instrument
[0148] The mean particle diameter (i.e., Peak 1/Peak 2) for these
microfluidized plant sterol nanoemulsions was 39 nm. See FIG. 1.
The average particle diameter data for the plant sterol
microfluidized nanoemulsion is shown in Table 2 below.
TABLE-US-00002 TABLE 2 Microfluidized Plant Sterol Nanoemulsion
Diam. (nm) % Intensity Width (nm) Peak 1: 54.16 85.86 14.36 Peak 2:
15.55 14.14 2.521 Peak 3: 0 0 0 Z-Average: 38.91; PDI: 0.228;
Intercept: 0.9764.
[0149] After three months the particle diameter was again
determined. The mean particle diameter (i.e., Peak 1) for this
microfluidized plant sterol nanoemulsion was 64.4 nm. See FIG. 1A.
The average particle diameter data for the three month plant sterol
nanoemulsion is shown in Table 3 below.
TABLE-US-00003 TABLE 3 Three Month Storage: Microfluidized Plant
Sterol Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1: 74.8
100 120.8 Peak 2: 0 0 0 Peak 3: 0 0 0 Z-Average: 64.4; PDI: 0.196;
Intercept: 0.969.
Example 2
Stable Formulation of Cod Liver Oil Microfluidized
Nanoemulsions
[0150] This example presents one cod liver oil embodiment of a
microfluidized nanoemulsion that has a stable particle diameter for
at least four months. The step-wise procedure is as follows:
[0151] 1. Heat 5 g of soybean oil (65.degree. C.)
[0152] 2. Add 5 g cod liver oil, stir and heat to 80.degree. C.
[0153] 3. Add 6 g polysorbate 80, stir and heat 20 mins
[0154] 4. Add 200 mL de-ionized water, stir and heat 30 mins
[0155] 5. Microfluidize using a M-110EH unit once at 25,000 PSI
[0156] 6. Do particle diameter analysis using a Malvern Nano S
instrument
[0157] The mean particle diameter (i.e., Peak 1/Peak 2) for this
cod liver oil microfluidized nanoemulsion was 58 nm. Before
microfluidization, the mean particle diameter of the cod liver oil
suspension was 2,842 nm. This represents a 50-fold reduction with a
single pass through the microfluidizer. Four months after the
microfluidization process, the particle diameter was again
determined and found not to have changed. See FIG. 2. The average
particle diameter data from the four-month microfluidized sample is
presented in Table 4.
TABLE-US-00004 TABLE 4 Microfluidized Cod Liver Oil Nanoemulsion
Four Months After Preparation Diam. (nm) % Intensity Width(nm) Peak
1: 63.92 82.22 15.62 Peak 2: 18.51 17.78 2.771 Peak 3: 0 0 0
Z-Average: 45.15; PDI: 0.247; Intercept: 0.9707.
Example 3
Stable Formulation of Tocopherol Microfluidized Nanoemulsions
[0158] This example presents one tocopherol embodiment of a
microfluidized nanoemulsion that maintains particle diameter for at
least five months. The step-wise procedure is as follows:
[0159] 1. Heat 13.5 g of soybean oil
[0160] 2. Add 2 g tocopherol, stir and heat to 90.degree. C.
[0161] 3. Heat 2 g polysorbate 80 in 100 mL de-ionized water, heat
to 75.degree. C.
[0162] 4. Add step 3 mixture to step 2 mixture
[0163] 5. Heat 300 mL di-ionized water and 6 g polysorbate 80, heat
till 70.degree. C.
[0164] 6. Add step 4 mixture to step 5 mixture, keep stir bar and
heat on
[0165] 7. Homogenize step 6 mixture for 2-4 mins
[0166] 8. Stir formulation for 3-5 mins on hot plate
[0167] 9. Microfluidize using a M-110EH unit once at 25,000 PSI
[0168] 10. Do particle diameter analysis using a Malvern Nano S
instrument
[0169] The mean particle diameter for the tocopherol microfluidized
nanoemulsion was 64 nm. Before microfluidization, the mean particle
diameter for the tocopherol suspension was 1,362 nm. This
represents a 21-fold reduction a single pass through the
microfluidizer. Five months after the microfluidization process,
the particle diameter was again determined and found not to have
changed. See FIG. 3. The average particle diameter data from the
five-month microfluidized sample is presented in Table 5.
TABLE-US-00005 TABLE 5 Microfluidized Tocopherol Nanoemulsion Five
Months After Preparation Diam. (nm) % Intensity Width (nm) Peak 1
88.06 77.84 19.99 Peak 2 26.46 22.16 3.651 Peak 3 0 0 0 Z-Average:
58.07; PDI: 0.234; Intercept: 0.9697
Example 4
Formulation of Lutein and Zeaxanthin Microfluidized
Nanoemulsions
[0170] This example presents one lutein/zeaxanthin embodiment of a
microfluidized nanoemulsion. The step-wise procedure is as
follows:
[0171] 1. Heat 5 g of soybean oil
[0172] 2. Add 2 g of lecithin
[0173] 3. Heat and stir, 10 mins
[0174] 4. Add 125 mg of lutein and 125 mg of zeaxanthin
[0175] 5. Heat and stir, 10 mins
[0176] 6. Heat 240 ml de-ionized water, 50.degree. C.
[0177] 7. Add heated water to mixture
[0178] 8. Stir and heat, till it is a solution
[0179] 9. Microfluidize using a M-110EH unit once at 25,000 PSI
[0180] 10. Do particle diameter analysis using a Malvern Nano S
instrument
[0181] The mean particle diameter (i.e., Peak 1/Peak 2) for the
lutein and zeaxanthin microfluidized nanoemulsion was 62 nm. See
FIG. 4. The average particle diameter data for the sample is shown
in Table 6.
TABLE-US-00006 TABLE 6 Microfluidized Lutein/Zeaxanthin
Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1 89.45 83.96
21.1 Peak 2 22.81 16.04 2.968 Peak 3 0 0 0 Z-Average: 62.26 PDI:
0.245 Intercept: 0.976
Example 5
Formulation of Soy Protein Microfluidized Nanoemulsion
[0182] This example presents one soy protein embodiment of a
microfluidized nanoemulsion. The step-wise procedure is as
follows:
[0183] 1. Heat 5 g soybean oil
[0184] 2. Add 5 g liquid lecithin
[0185] 3. Heat and stir 10 mins, 70.degree. C.
[0186] 4. Heat 240 mL de-ionized water, 65.degree. C.
[0187] 5. Add heated water to mixture
[0188] 6. Add 9 g soy protein, stir and heat 10 min
[0189] 7. Add 9 g soy protein
[0190] 8. Stir and heat 20 min, 70.degree. C.
[0191] 9. Homogenize 1 min
[0192] 10. Microfluidize using a M-110EH unit ten times at 25,000
PSI
[0193] 11. Do particle diameter analysis using a Malvern Nano S
instrument
[0194] The mean particle diameter (i.e., Peak 1/Peak 2) for the
vanilla soy protein (Central Soya) microfluidized nanoemulsion was
55 nm. See FIG. 5. The average particle diameter data for the
sample is shown in Table 7.
TABLE-US-00007 TABLE 7 Microfluidized Soy Protein Nanoemulsion
Diam. (nm) % Intensity Width (nm) Peak 1 55.15 80.32 16.45 Peak 2
290.8 19.68 82.82 Peak 3 0 0 0 Z-Average: 54.97; PDI: 0.283;
Intercept: 0.9819
Example 6
Formulation of Whey Protein Microfluidized Nanoemulsion
[0195] This example presents one whey protein embodiment of a
microfluidized nanoemulsion. The step-wise procedure is as
follows:
[0196] 1, Heat 5 g soybean oil
[0197] 2. Add 5 g soy lecithin
[0198] 3. Add 250 mg polysorbate 80
[0199] 4. Heat and stir 10 mins, 70.degree. C.
[0200] 5. Heat 240 mL de-ionized water, 65.degree. C.
[0201] 6. Add heated water to mixture
[0202] 7. Add 10 g whey protein
[0203] 8. Stir and heat 10 min
[0204] 9. Homogenize 1 min
[0205] 10. Microfluidize using a M-110EH unit once at 25,000
PSI
[0206] 11. Do particle diameter analysis Malvern Nano S
instrument
[0207] The mean particle diameter (i.e., Peak 1/Peak 2) for the
whey protein microfluidized nanoemulsion was 108 nm. See FIG. 6.
The average particle diameter data for the sample is shown in Table
8.
TABLE-US-00008 TABLE 8 Microfluidized Whey Protein Nanoemulsion
Diam. (nm) % Intensity Width (nm) Peak 1 127.7 91.3 38.09 Peak 2
23.72 6.161 2.764 Peak 3 5027 2.536 593 Z-Average: 108.2 PDI: 0.263
Intercept: 0.948
Example 7
Formulation of Orange Juice, Plant Sterol and Lutein Microfluidized
Nanoemulsion
[0208] This example presents one orange juice/plant sterol/lutein
embodiment of a microfluidized nanoemulsion. The step-wise
procedure is as follows:
[0209] 1. Heat soybean oil, 80.degree. C.
[0210] 2. Add 1.5 g plant sterol
[0211] 3. Stir and heat, 5 min
[0212] 4. Add 5 g polysorbate 80
[0213] 5. Add 70 mg Lutein
[0214] 6. Stir and heat, 10 min
[0215] 7. Add 240 mL orange juice (Tropicana.RTM.)
[0216] 8. Stir and heat, 1 hour
[0217] 9. Microfluidize using a M-110EH unit twice at 25,000
PSI
[0218] 10. Do particle diameter analysis using a Malvern Nano S
instrument
[0219] The mean particle diameter (i.e., Peak 1/Peak 2) for the
orange juice/plant sterol/lutein microfluidized nanoemulsion was 46
nm. See FIG. 7. The average particle diameter data for the sample
is shown in Table 9.
TABLE-US-00009 TABLE 9 Microfluidized Orange Juice/Plant
Sterol/Lutein Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1
61.55 81.57 15.32 Peak 2 17.13 16.1 2.433 Peak 3 5143 2.329 509.4
Z-Average: 46.41; PDI: 0.322; Intercept: 0.9609
Example 8
Stable Formulation of DHA Fish Oil/Water Microfluidized
Nanoemulsion
[0220] This example presents one DHA fish oil/water embodiment of a
microfluidized nanoemulsions that maintains particle diameter for
at least two months. The step-wise procedure is as follows:
[0221] 1. Heat 6.4 g DHA fish oil
[0222] 2. Add 6 g soy lecithin
[0223] 3. Add 250 mg polysorbate 80
[0224] 4. Heat 240 mL de-ionized water, 75.degree. C.
[0225] 5. Add heated water to mixture
[0226] 6. Stir and heat, 20 mins
[0227] 7. Homogenize 2 mins
[0228] 8. Stir and heat, 10 mins
[0229] 9. Microfluidize using a M-110EH unit once at 25,000 PSI
[0230] 10. Do particle diameter analysis using a Malvern Nano S
instrument.
[0231] The mean particle diameter (i.e., Peak 1) for the DHA fish
oil/water microfluidized nanoemulsion was 73 nm. Two months after
the microfluidization process, the particle diameter was again
determined and found not to have changed. See FIG. 8. The average
particle diameter data from the two-month microfluidized sample is
presented in Table 10.
TABLE-US-00010 TABLE 10 Stable Microfluidized DHA Fish Oil/Water
Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1 81.73 100
20.38 Peak 2 0 0 0 Peak 3 0 0 0 Z-Average: 72.58; PDI: 0.205;
Intercept: 0.9636.
Example 9
Stable Formulation of DHA Fish Oil/Milk Microfluidized
Nanoemulsion
[0232] This example presents one DNA fish oil/milk embodiment
without any added emulsifiers that maintains particle diameter for
at least three (3) weeks. The step-wise procedure is as
follows:
[0233] 1. Heat 1.5 g DHA fish oil, 50.degree. C.
[0234] 2. Heat 200 mL whole milk, 50.degree. C.
[0235] 3. Mix the two together
[0236] 4. Stir and heat, 10 mins
[0237] 5. Microfluidize using a M-110EH unit once at 25,000 PSI
[0238] 6. Do particle diameter analysis using a Malvern Nano S
instrument
[0239] The mean particle diameter (i.e., Peak 1) for the DHA fish
oil/milk microfluidized nanoemulsion 93 nm. This nano-emulsion
preparation was made without any added emulsifiers. Three weeks
after the microfluidization process, the fish oil was still in
solution and the particle diameter was again determined and found
not to have changed. See FIG. 9. The average particle diameter data
from the three-week microfluidized sample is presented in Table
11.
TABLE-US-00011 TABLE 11 Stable Microfluidized DNA Fish Oil/Milk
Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1 106.9 100
32.84 Peak 2 0 0 0 Peak 3 0 0 0 Z-Average: 93.11; PDI: 0.178;
Intercept: 0.9341
Example 10
Temperature Stability of Microfluidized Nanoemulsions
[0240] This example presents the stability of microfluidized
nanoemulsions following exposure to either heat or cold. The
formulation used in this experiment comprised DHA Fish Oil
milk/tocopherol.
[0241] 1. Dissolved 1 g of vitamin C in 25 mL of di-ionized
water
[0242] 2. Added 200 mL of whole milk to step 1
[0243] 3. Took 1.7 g DHA fish oil and added 800 mg of delta
tocopherol
[0244] 4. Added steps 1 and 2 to step 3
[0245] 5. Stir and heat 10 mins, 50.degree. C.
[0246] 6. Microfluidize using a M-110EH unit once at 25,000 PSI
[0247] 7. Do particle diameter analysis using a Malvern Nano S
instrument
[0248] The mean particle diameter (i.e., Peak 1) for the DHA fish
oil/milk/tocopherol microfluidized nanoemulsion was 87 nm. See FIG.
10. This nano-emulsion preparation was made without any added
emulsifiers. The average particle diameter data for the original
nanoemulsion is presented in Table 12.
TABLE-US-00012 TABLE 12 Microfluidized DNA Fish Oil/Milk/Tocopherol
Original Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1
91.84 97.86 23.95 Peak 2 5179 2.144 485.1 Peak 3 0 0 0 Z-Average:
87.09; PDI: 0.216; Intercept: 0.9339
[0249] This original microfluidized nanoemulsion was pasteurized at
75.degree. C. for 30 seconds. Twenty-four hours later, the oil was
still in solution and the particle diameter was stable as compared
to the original nanoemulsion. See FIG. 11. The average particle
diameter data for the pasteurized microfluidized nanoemulsion is
presented in Table 13.
TABLE-US-00013 TABLE 13 Microfluidized DHA Fish Oil/Milk/Tocopherol
Pasteurized Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1
108.3 82.49 28.06 Peak 2 45.16 17.51 8.109 Peak 3 0 0 0 Z-Average:
87.18; PDI: 0.198; Intercept: 0.9281
[0250] The original microfluidized nanoemulsion was freeze-thaw
tested at -4.degree. C. for 24 hours. Twenty-four hours later, the
oil was still in solution and the particle diameter was stable as
compared to the original nanoemulsion. See FIG. 12. The average
particle diameter data for the freeze-thaw microfluidized
nanoemulsion is presented in Table 14.
TABLE-US-00014 TABLE 14 Microfluidized DHA Fish Oil/Milk/Tocopherol
Freeze-Thaw Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1
99.72 100 39.07 Peak 2 0 0 0 Z-Average: 87.58; PDI: 0.198
Example 11
Improved Bioavailability of Dietary Lycopene
[0251] This example demonstrates an improved bioavailability of
lycopene when fed as a uniform microfluidized nanoemulsion versus
mixed into a standard diet formulation.
[0252] The lycopene microfluidized nanoemulsion was prepared in a
step-wise manner as follows:
[0253] 1. Heat 5 g of soybean oil
[0254] 2. Add 2 g of lecithin
[0255] 3. Heat and stir, 10 mins
[0256] 4. Add 125 mg of lycopene
[0257] 5. Heat and stir, 10 mins
[0258] 6. Heat 240 ml de-ionized water (or grape juice); 50.degree.
C.
[0259] 7. Add heated water (or grape juice) to mixture
[0260] 8. Stir and heat, till it is a solution
[0261] 9. Microfluidize using a M-110EH unit once at 25,000 PSI
[0262] 10. Do particle diameter analysis using a Malvern Nano S
instrument
[0263] The mean particle diameter for the lycopene microfluidized
nanoemulsion was 74 nm.
Bioavailability In Gerbils
[0264] The microfluidized nanoemulsion was incorporated into a
chow-based diet and fed to gerbils over a 4 week period. A control
group was fed a lycopene in oil-enriched chow-based diet. At the
end of 4 weeks, blood was collected, plasma harvested and plasma
lycopene levels were determined by HPLC in both gerbil groups.
[0265] FIG. 13 demonstrates that control gerbils did not
demonstrate detectable plasma lycopene levels. The gerbils fed a
chow comprising a microfluidized lycopene nanoemulsion, however,
demonstrated elevated plasma lycopene levels. See FIG. 14,
Bioavailability In Humans
[0266] A microfluidized lycopene nanoemulsion was then prepared
with grape juice instead of water and orally administered to two
(2) human subjects over a 4 day period (125 mg/serving, 2 servings
per day). This administration raised plasma lycopene levels by
approximately 38% (data not shown).
Example 12
Improved Efficacy of Microfluidized Nanoemulsions
[0267] This example presenting data showing that microfluidized
nanoemulsions provide improved efficacy over that seen in
traditional nanoemulsions. Specifically, this example compares the
ability of three plant sterol formulations to reduce plasma low
density lipoprotein cholesterol (LDL-C) levels in
hypercholesterolemic hamsters.
[0268] A microfluidized mixed plant sterol (60% sitosterol)
nanoemulsion was prepared in a step-wise manner as follows:
[0269] 1. Heat 5 g soybean oil.
[0270] 2. Add 5 g soy lecithin, stir and heat 15 mins.
[0271] 3. Repeat Step 2.
[0272] 4. Add 15 g soybean oil, stir and heat 10 mins.
[0273] 5. Add 4 g plant sterol, stir and heat 10 mins.
[0274] 6. Repeat Step 4 four (4) times.
[0275] 7. Add 1 g polysorbate 80, stir and heat 10 mins.
[0276] 8. Heat 200 ml MinuteMaid Heartwise.RTM. orange juice
(75.degree. C.).
[0277] 10. Heat 1800 ml MinuteMaid Heartwise.RTM. orange juice
(70.degree. C.).
[0278] 11. Add Step 8 to Step 7. Stir and heat 20 min (80.degree.
C.).
[0279] 12. Add to Step 10.
[0280] 13. Add 1 g polysorbate 80, stir and heat 20 min (80.degree.
C.).
[0281] 14. Homogenize for 2-4 min.
[0282] 15. Stir homogenate on hot plate for 10 min.
[0283] 16. Microfluidize using a M-110EH unit at 25,000 PSI.
[0284] 17. Do particle analysis using a Malvern Nano S
instrument.
[0285] The mean particle diameter for the microfluidized plant
sterol nanoemulsion was 41.95 nm. See FIG. 16.
[0286] Forty (40) hamsters were divided into four (4) groups of ten
(10) each. Group I was fed a control hypercholesterolemic diet
(HCD); Group 2 was fed 30 mg/d of crystalline plant sterol; Group 3
was fed 20 mg/d of MinuteMaid Heartwise.RTM. micronized plant
sterol (Cargill); Group 4 was fed 10 mg/d of the microfluidized
plant sterol nanoemulsion. After four (4) weeks, blood samples were
analyzed for plasma LDL-C levels. The microfluidized plant sterol
nanoemulsion was twice as effective as the MinuteMaid
Heartwise.RTM. micronized diet, and three times as effective as the
crystalline plant sterol diet. See FIG. 17.
[0287] The data show that the improved bioavailability shown in
Example 11 results in improved clinical therapy when compared to
micron-sized or crystalline plant sterol diets.
Example 13
Cholesterol Nanoemulsions
Insoluble vs Soluble Dispersion Media
[0288] This example presents data demonstrating that uniform
microfluidized nanoemulsion compositions depend upon a compound
having substantial solubility in the liquid dispersion medium. This
example compares the microfluidizing technique described in U.S.
Pat. No. 5,510,118 to one embodiment as contemplated by the present
invention. The absorbable lipid cholesterol was chosen as the test
compound.
[0289] Group I represents the '118 premix and was prepared by
dispersing cholesterol (2 gms), water (100 mls) and Tween.RTM. 80
(0.2 gms), where cholesterol is insoluble (i.e., below at least 30
mg/ml) in the liquid dispersion medium (water). Thereafter, this
cholesterol/water/Tween.RTM. 80 solution was microfluidized using a
M-100EH unit. Multiple passes (10-15) through the microfluidizer
were performed at PSI's ranging between 4,000-20,000 but were
terminated because the generated heat exceeded 70.degree. C. (much
higher than the recommended 30-40.degree. C. in the '118 patent.
After the microfluidization it was observed that much of the
cholesterol had precipitated. After twenty-four hours, the
preparation of the Group I nanoemulsion contained only 0.44 gms
(i.e., 22%) of the original cholesterol weight.
[0290] Group II represents one embodiment of the present invention
and was prepared by dispersing cholesterol (2 gms) in heated
soybean oil (10 gms), soy lecithin (5 gms), and Tween.RTM. 80 (0.2
gms) where cholesterol is substantially soluble (i.e., above at
least 30 mg/ml) in the dispersion medium (oil). Thereafter, this
cholesterol/oil/lecithin/Tween.RTM. 80 was added to 100 ml of
heated water and microfluidized using a single 30 second pass at
25,000 PSI using a M-100EH unit. After the microfluidization
cholesterol precipitation was not noticeably evident. After
twenty-four hours, the preparation of the Group II nanoemulsion
contained 1.66 gm (i.e., 83%) of the original cholesterol
weight.
[0291] The data show that the particle diameter distributions from
both Group I and Group II premix preparations are practically
identical. See FIG. 18A and FIG. 18B, respectively. Specifically, a
single peak ranging from 700-1000 nm having a mean particle
diameter of approximately 900 nm is observed for both preparations.
See Tables 15 and 16.
TABLE-US-00015 TABLE 15 Cholesterol/Tween .RTM. 80/Water Premix
Particle diameter: Group I Diam. (nm) % Intensity Width (nm) Peak 1
942.5 100 38.9 Z-Average: 1982; PDI: 0.210; Intercept: 0.6797
TABLE-US-00016 TABLE 16 Cholesterol/Oil/Lecithin/Tween .RTM.
80/Water Premix Particle diameter: Group II Diam. (nm) % Intensity
Width (nm) Peak 1 897.9 100 64.8 Z-Average: 1328; PDI: 0.427;
Intercept: 0.6989
[0292] Following microfluidization, however, the particle diameter
distributions are vastly different between Group I and Group II.
See FIG. 19A and FIG. 19B, respectively. Group I shows two vastly
disparate and distinct peaks. See Table 17. Group II, however,
shows a single peak representing one embodiment of a uniform
microfluidized nanoemulsion. See Table 18.
TABLE-US-00017 TABLE 17 Microfluidized Cholesterol/Tween .RTM.
80/Water Nanoemulsion Diam. (nm) % Intensity Width (nm) Peak 1
578.2 67.6 120.8 Peak 2 96.7 32.3 14.9 Z-Average: 246.5; PDI:
0.789; Intercept: 0.7687
TABLE-US-00018 TABLE 18 Microfluidized
Cholesterol/Oil/Lecithin/Tween .RTM. 80/Water Nanoemulsion Diam.
(nm) % Intensity Width (nm) Peak 1 101.3 100 25.1 Z-Average: 86.8;
PDI: 0.240; Intercept: 0.9455
[0293] The data above demonstrate that some embodiments of the
present invention contemplate improvements over the art in creating
uniform microfluidized nanoemulsions. In particular, it is now
clear that the Bosch et al ('118 patent), and the Cooper et al.
portfolio ('758, '038, and '202 application publications) do not
teach a microfluidization process that creates a uniform particle
diameter distribution.
Example 14
Nanoparticulate Compositions vs Uniform Microfluidized
Nanoemulsions
[0294] This example describes a demonstration that will show that a
milled nanoparticle composition (for example, one made according to
US Appln Publ No. 2004/0033202 to Cooper et al.) does not create a
uniform particle diameter distribution as does a microfluidized
nanoemulsion as contemplated by one embodiment of the present
invention. An absorbable phytosterol will be chosen as the test
compound.
[0295] Group I represents the '202 premix that will be prepared by
dispersing 5% (w/w) phytosterol/water solution with 1% (w/w)
Tween.RTM. 80, where the phytocholesterol is insoluble (i.e., below
at least 30 mg/ml) in the liquid dispersion medium (water).
Thereafter, this phytosterol/water/Tween.RTM. 80 solution will be
milled at 10.degree. C. for 1.5 to 2 hours in a DYNO.RTM.-Mill KDL
(Willy A Bachofen A G, Machinefabrik, Basel, Switzerland) using a
500 .mu.m milling media (i.e., grinding beads) of type
Polymill.RTM. 500. After the milling it will be observed that much
of the phytocholesterol has precipitated. After at least
twenty-four hours, the preparation of the Group I nanoparticulate
will contain less than 1/2 of the original phytosterol weight.
[0296] Group II represents one embodiment of the present invention
and will be prepared by dispersing 5% (w/w) phytosterol/heated
soybean oil solution, soy lecithin, with 1% Tween.RTM. 80, where
the phytosterol is substantially soluble (i.e., above at least 30
mg/ml) in the liquid dispersion medium (oil). Thereafter, this
phytosterol/oil/lecithin/Tween.RTM. 80 premix is added to 100 ml
heated water and microfluidized using a single 30 second pass at
25,000 PSI using a M-100EH unit. After the microfluidization
phytosterol precipitation will not be noticeably evident. After
twenty-four hours, the preparation of the Group II nanoemulsion
will contain greater than 3/4 of the original phytosterol
weight.
[0297] The data will show that the particle diameter distributions
from both Group I and Group II premix preparations are practically
identical. For example, a single peak ranging from 700-1000 nm
having a mean particle diameter of approximately 900 nm might be
observed for both preparations. See Tables 19 and 20.
TABLE-US-00019 TABLE 19 Phytosterol/Tween 80/Water Premix Particle
diameter: Group I Diam. (nm) % Intensity Width (nm) Peak 1 942.5
100 38.9 Z-Average: 1982; PDI: 0.210; Intercept: 0.6797
TABLE-US-00020 TABLE 20 Phytosterol/Oil/Lecithin/Tween 80/Water
Premix Particle diameter: Group II Diam. (nm) % Intensity Width
(nm) Peak 1 897.9 100 64.8 Z-Average: 1328; PDI: 0.427; Intercept:
0.6989
[0298] Following processing however, the particle diameter
distributions are expected to be vastly different between Group I
and Group II. For example, Group I will most likely show at least
two vastly disparate and distinct peaks. See Table 21. Group II,
however, will have only a single peak representing one embodiment
of a uniform microfluidized nanoemulsion. See Table 22.
TABLE-US-00021 TABLE 21 Microfluidized Cholesterol/Tween 80/Water
Nanoemulsion: Group I Diam. (nm) % Intensity Width (nm) Peak 1
578.2 67.6 120.8 Peak 2 96.7 32.3 14.9 Z-Average: 246.5; PDI:
0.789; Intercept: 0.7687
TABLE-US-00022 TABLE 22 Microfluidized
Cholesterol/Oil/Lecithin/Tween 80/Water Nanoemulsion: Group II
Diam. (nm) % Intensity Width (nm) Peak 1 101.3 100 25.1 Z-Average:
86.8; PDI: 0.240; Intercept: 0.9455
[0299] The data above demonstrate that nanoparticulate composition
are not able to create uniform particle diameter distributions as
contemplated by some embodiments of the nanoemulsions contemplated
herein. In particular, it is now clear that the Cooper et al.
portfolio ('758, '038, and '202 application publications) do not
teach a milling process that creates a uniform particle diameter
distribution.
Example 15
Improved Bioavailability Over Conventional Nanoparticulate
Compositions
[0300] This example will provide data showing that a uniform
microfluidized nanoemulsion as contemplated by one embodiment of
the present invention has improved plant sterol bioavailability
and/or efficacy than a conventional nanoparticulate
composition.
[0301] A standard curve will be constructed by gavaging thirty (30)
hamsters with 1 .mu.Ci .sup.3H-cholesterol. Plasma cholesterol
levels are then determined at Day 1, Day 2, Day 4, and Day 7. These
data are used to calculate bioavailability of .sup.3H-cholesterol
during the 7 Day period as area-under-the-curve (AUC).
[0302] After plasma radioactivity levels have returned to
background levels (i.e., approximately 7.5 cholesterol metabolic
half-lives), the experiment will be repeated using the following
treatment groups (n=10). [0303] Group I: Standard diet mixed with a
plant sterol. [0304] Group II: Standard diet mixed with a uniform
microfluidized plant sterol nanoemulsion prepared in accordance
with Example 1. [0305] Group III: Standard diet mixed with a
conventional lycopene nanoparticulate composition prepared in
accordance with conventional milling grinder techniques as
described in the '202 Cooper et al. application.
[0306] The AUC measurement will determine the ability of each
preparation to reduce the absorption of .sup.3H-cholesterol into
the bloodstream which is proportional to the bioavailability and/or
efficacy of each preparation.
[0307] A greater bioavailability and/or efficacy of a plant sterol
when administered as a uniform microfluidized nanoemulsion will be
seen because: i) the average particle diameter of the uniform
microfluidized nanoemulsion is smaller than the conventional
nanoparticulate composition (i.e., for example, 300 nm v. 50 nm);
ii) microfluidization produces more stable particles than either
milling or homogenization; and iii) microfluidization produces
pH-resistant particles (i.e., stomach acid or small intestine base
conditions) unlike those produced by either milling or
homogenization.
Example 16
Improved Efficacy Over Conventional Nanoparticulate
Compositions
[0308] This example will provide data showing that a uniform
microfluidized nanoemulsion as contemplated by one embodiment of
the present invention has improved efficacy in lowering plasma
cholesterol levels that a conventional nanoparticulate
composition.
[0309] The study will have duration of six (6) weeks. Briefly,
seventy (70) hamsters will be fed a liquid-based
hypercholesterolemic diet for a two (2) week pre-test period in
order to elevate and stabilize plasma cholesterol levels.
Subsequently, the hamsters are divided into the seven (7) test
groups (n=10) shown below. Each group is maintained on the
liquid-based hypercholesterolemic diet and: i) a nanoparticulate
composition (i.e., for example, prepared as per the '202 Cooper et
al. application); or ii) a uniform microfluidized nanoemulsion as
contemplated by one embodiment of the present invention, for four
(4) additional weeks. [0310] Group 1: Hypercholesterolemic diet
only [0311] Group II: Hypercholesterolemic diet+0.1% (w/w) plant
sterol nanoparticulate composition. [0312] Group III:
Hypercholesterolemic diet+0.5% (w/w) plant sterol nanoparticulate
composition. [0313] Group IV: Hypercholesterolemic diet+1% (w/w)
plant sterol nanoparticulate composition. [0314] Group V:
Hypercholesterolemic diet+0.1% (w/w) plant sterol uniform
microfluidized nanoemulsion. [0315] Group VI: Hypercholesterolemic
diet+0.5% (w/w) plant sterol uniform microfluidized nanoemulsion.
[0316] Group VII: Hypercholesterolemic diet+1% (w/w) plant sterol
uniform microfluidized nanoemulsion.
[0317] Blood samples are taken at 0, 2, 3, 4, 5, and 6 weeks where
plasma cholesterol levels will be determined by methods known in
the art.
[0318] A greater efficacy of the plant sterol uniform
microfluidized nanoemulsions to lower plasma cholesterol levels is
seen because: i) the average particle diameter of the uniform
microfluidized nanoemulsion is smaller than the conventional
nanoparticulate composition (i.e., for example, 300 nm v. 50 nm);
ii) microfluidization produces more stable particles than either
milling or homogenization; and iii) microfluidization produces
pH-resistant particles (i.e., stomach acid or small intestine base
conditions) unlike those produced by either milling or
homogenization.
Example 17
Microfluidization Single Pass Comparison
[0319] This example provides data showing that the Bosch technique
does not produce a uniform microfluidized nanoemulsion when
compared to one embodiment of the present invention under identical
microfluidization techniques.
[0320] The Group I & II premixes were prepared in accordance
with Example 13. Each premix was subjected to one pass at 25,000
PSI in the microfluidizer. Group I (representing the Bosch
formulation) shows that 85% of the particles have a mean diameter
of 815 nm. See FIG. 20A. Group II (representing one embodiment of
the present invention) shows that 98% of the particles have a mean
diameter of 78 nm, See FIG. 20B This represents a greater than
ten-fold difference in average diameter. Significantly, only 15% of
the Bosch particles are within the 100 nm range, thereby
representing a six-fold difference in particle diameter
distribution in this lower range.
[0321] The average particle diameter distributions between Group I
and Group II are presented in Tables 23 & 24 below.
TABLE-US-00023 TABLE 23 Microfluidized Cholesterol/Tween .RTM.
80/Water Nanoemulsion: Single Pass Diam. (nm) % Intensity Width
(nm) Peak 1 815.3 84.5 117.7 Peak 2 101.8 15.54 10.54 Z-Average:
651.5; PDI: 84.5; Intercept: 0.7487
TABLE-US-00024 TABLE 24 Microfluidized
Cholesterol/Oil/Lecithin/Tween .RTM. 80/Water Nanoemulsion: Single
Pass Diam. (nm) % Intensity Width (nm) Peak 1 78.43 97.47 31.43
Peak 2 19.63 2.535 2.928 Z-Average: 65.98; PDI: 0.190; Intercept:
0.9210
* * * * *