U.S. patent application number 14/835388 was filed with the patent office on 2016-02-25 for egg protein formulations and methods of manufacture thereof.
The applicant listed for this patent is Aimmune Therapeutics, Inc.. Invention is credited to Howard V. Raff.
Application Number | 20160051639 14/835388 |
Document ID | / |
Family ID | 55347334 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160051639 |
Kind Code |
A1 |
Raff; Howard V. |
February 25, 2016 |
EGG PROTEIN FORMULATIONS AND METHODS OF MANUFACTURE THEREOF
Abstract
The present technology relates generally to formulations
comprising egg white protein, methods of manufacturing egg protein
formulations and uses for egg protein formulations. In particular,
several embodiments are directed to egg protein formulations for
oral administration in immunotherapy of subjects affected by egg
allergies.
Inventors: |
Raff; Howard V.; (Mill
Valley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aimmune Therapeutics, Inc. |
Brisbane |
CA |
US |
|
|
Family ID: |
55347334 |
Appl. No.: |
14/835388 |
Filed: |
August 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62041362 |
Aug 25, 2014 |
|
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|
Current U.S.
Class: |
424/94.61 |
Current CPC
Class: |
A61P 37/08 20180101;
C12Y 302/01017 20130101; A61K 9/4866 20130101; A61K 38/38 20130101;
A61K 38/1735 20130101; A61K 35/57 20130101; A61K 38/55 20130101;
A61K 38/47 20130101 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 38/38 20060101 A61K038/38; A61K 38/17 20060101
A61K038/17 |
Claims
1. A method of manufacturing a low dose egg protein formulation,
comprising: a) mixing an amount of egg white powder comprising egg
white protein having characterized ovomucoid, ovalbumin, and
lysozyme proteins and a diluent in a first blend; b) adding between
50-99% diluent in a second blend; c) adding one or more of a
diluent, filling agent, lubricant or glidant in a final blend; and
d) providing the blended powder in a capsule or a pouch.
2. The method of claim 1, wherein the dose of egg white protein per
capsule is about 0.2 mg egg white protein.
3. The method of claim 1, wherein the dose of egg white protein per
capsule is about 1.0 mg egg white protein.
4. The method of claim 1, wherein the dose of egg white protein per
capsule is about 10.0 mg egg white protein.
5. The method of claim 1, wherein the dose of egg white protein is
20 mg or more and the blended powder is provided in a capsule.
6. The method of claim 1, wherein the dose of egg white protein is
100 mg or more and the blended powder is provided in a pouch.
7. The method of claim 1, wherein the dose of egg white protein is
300 mg or more and the blended powder is provided in a pouch.
8. The method of claim 1, wherein the dose of egg white protein is
1000 mg or more and the blended powder is provided in a pouch.
9. The method of claim 1, wherein levels of egg white protein in
the egg white protein powder are stable in the pharmaceutical
formulation for about 3, 6, 9, 11 or more months.
10. The method of claim 1, wherein a concentration of one or more
of ovomucoid, ovalbumin, and lysozyme proteins in the formulation
are stable for about 3, 6, 9, or 11 or more months.
11. The method of claim 1, wherein the diluent is selected from the
group consisting of alginic acid and salts thereof; cellulose
derivatives; silicified microcrystalline cellulose;
microcrystalline dextrose; amylose; magnesium aluminum silicate;
polysaccharide acids; bentonites; gelatin;
polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone;
povidone; starch; pregelatinized starch; tragacanth, dextrin, a
sugar; dicalcium phosphate; a natural or synthetic gum;
polyvinylpyrrolidone, larch arabogalactan, Veegum.RTM.,
polyethylene glycol, waxes, sodium alginate, a starch; a
cross-linked starch; a cross-linked polymer; a cross-linked
polyvinylpyrrolidone; alginate; a clay; a gum; sodium starch
glycolate; bentonite; a natural sponge; a surfactant; a resin;
citrus pulp; sodium lauryl sulfate; and sodium lauryl sulfate in
combination starch.
12. The method of claim 1, wherein the filling agent is selected
from the group consisting of lactose, calcium carbonate, calcium
phosphate, dibasic calcium phosphate, calcium sulfate,
microcrystalline cellulose, cellulose powder, dextrose, dextrates,
dextran, starches, pregelatinized starch, sucrose, xylitol,
lactitol, mannitol, sorbitol, sodium chloride, and polyethylene
glycol.
13. The method of claim 1, wherein the lubricant is selected from
the group consisting of stearic acid, calcium hydroxide, talc, corn
starch, sodium stearyl fumerate, alkali-metal and alkaline earth
metal salts, stearic acid, sodium stearates, magnesium stearate,
zinc stearate, waxes, Stearowet.RTM., boric acid, sodium benzoate,
sodium acetate, sodium chloride, leucine, a polyethylene glycol or
a methoxypolyethylene glycol, propylene glycol, sodium oleate,
glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, and
magnesium or sodium lauryl sulfate.
14. The method of claim 1, wherein the characterized ovomucoid,
ovalbumin, and lysozyme proteins are characterized by size
exclusion chromatography.
15. The method of claim 1, wherein the diluent is Prosolv.
16. The method of claim 1, wherein the diluent is a mixture of
Prosolv SMCC and Prosolv HD90.
17. The method of claim 1, wherein the filling agent is
mannitol.
18. The method of claim 1, wherein the lubricant is magnesium
stearate.
19. The method of claim 1, wherein the glidant is talc.
20. The method of claim 1, wherein step (d) further comprises
passing the blended material through a mesh screen.
21. A method of making a capsule formulation, comprising, (a)
mixing egg white protein powder and a diluent in a first blend; (b)
discharging the blended material; (c) passing the blended material
through a mesh screen and blending the screened material in a
second blend; (d) adding in a filling agent, glidant and/or diluent
in a third blend; and (e) encapsulating the blended powder.
22. The method of claim 21, further comprising sampling the blended
material of step (d) one or more times prior to encapsulation.
23. The method of claim 21, wherein the dose comprises from about
0.2 to about 1000 mg egg white protein.
24. The method of claim 21, wherein the mesh screen of step (c)
comprises a # 20 mesh screen.
25. The method of claim 21, wherein the egg white protein comprises
lysozyme, ovomucoid, and ovalbumin.
26. The method of claim 21, wherein the concentration of lysozyme,
ovomucoid and ovalbumin is characterized by Size exclusion
chromatography (SEC)-HPLC.
27. The method of claim 26, wherein the concentration is at least
an amount of a reference standard.
28. The method of claim 21, wherein the encapsulated formulation is
stable for at least about 3, 6, 9, 12, 24, or 36 months.
29. The method of claim 21, wherein the encapsulated formulation is
stable at a temperature from about 2.degree. C. to about 30.degree.
C.
30. The method of claim 21, wherein the capsule comprises
Hydroxypropyl Methyl Cellulose (HPMC).
31. The method of claim 21, further comprising storing the
formulation in a container.
32. The method of claim 31, wherein the container is a bottle.
33. The method of claim 32, wherein the bottle is a Hi-Density
Propyl Ethylene bottle.
34. The method of claim 31, wherein the container further comprises
a desiccant packet to control moisture content of the container.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/041,362, filed Aug. 25, 2014, the disclosure of
which is herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present technology relates generally to formulations
comprising egg protein, methods of manufacturing egg protein
formulations and uses for dried egg protein formulations. In
particular, several embodiments are directed to egg protein
formulations for oral administration in immunotherapy of subjects
affected by allergies.
BACKGROUND
[0003] Food allergies, or the body's immunological reaction to
allergenic epitopes from proteins in the food, can severely impact
the quality of life for both adults and children. Egg, milk, and
peanut are the greatest sources of allergic response in affected
individuals, and can account for approximately 80% of all food
allergy cases. The severity of allergic reactions can vary between
individuals and can range from between mild irritation to
anaphylaxis, which can be so severe as to be life threatening, and
egg allergy has an approximate prevalence of 2.6% by age 2.5 years.
The current therapy for egg allergy is to place the child on an
egg-free diet until the allergy is outgrown (approximately 30% of
children), or to maintain the person on an egg-free diet
indefinitely. However, because egg is prevalent in processed foods
and vaccines, compliance can be difficult and creates a constant
challenge for egg-allergic individuals and their caregivers.
[0004] Allergic reactions result when a subject's immune system
responds to a foreign substance (e.g., allergen). Typically, there
is no allergic reaction the first time a subject is exposed to a
particular allergen. However, it is the initial response to an
allergen that primes the system for subsequent allergic reactions.
In particular, the allergen is taken up by antigen presenting cells
(APCs; e.g., macrophages and dendritic cells) that degrade the
allergen and then display allergen fragments to T-cells. T-cells,
in particular CD4+ "helper" T-cells, respond by secreting a
collection of cytokines that have effects on other immune system
cells. The profile of cytokines secreted by responding CD4+ T-cells
determines whether subsequent exposures to the allergen will induce
allergic reactions. Two classes of CD4+ T-cells (Th1 and Th2;
T-lymphocyte helper type) influence the type of immune response
that is mounted against an allergen.
[0005] The Th1-type immune response involves the stimulation of
cellular immunity to allergens and infectious agents and is
characterized by the secretion of IL-2, IL-6, IL-12, IFN-gamma, and
TNF-beta by CD4+ T helper cells and the production of IgG
antibodies. Exposure of CD4+ T-cells to allergens can also activate
the cells to develop into Th2 cells, which secrete IL-4, IL-5,
IL-10, and IL-13. IL-4 production stimulates maturation of B cells
that produce IgE antibodies specific for the allergen. These
allergen-specific IgE antibodies attach to mast cell and basophil
receptors, where they initiate a rapid immune response to the next
exposure to allergen. When the subject encounters the allergen a
second time, the allergen is quickly bound by these
surface-associated IgE molecules, resulting in the release of
histamines and other substances that trigger allergic reactions.
Subjects with high levels of IgE antibodies are known to be
particularly prone to allergies.
SUMMARY
[0006] Provided herein are egg protein formulations for use in the
treatment of egg allergy. Also provided herein is a method for
identifying and manufacturing egg protein formulations.
[0007] Provided herein is a method of manufacturing a low dose egg
protein formulation, by a) mixing an amount of egg white powder
comprising egg white protein having characterized ovomucoid,
ovalbumin, and lysozyme proteins and a diluent in a first blend; b)
adding between 50-99% diluent in a second blend; c) adding one or
more of a diluent, filling agent, lubricant or glidant in a final
blend; and d) providing the blended powder in a capsule or a pouch.
In some embodiments, step (d) of the method further comprises
passing the blended material through a mesh screen. In some
embodiments, the method further comprises characterizing the
ovomucoid, ovalbumin, and lysozyme proteins, for example, by size
exclusion chromatography. In some embodiments, step (d) of the
method further comprises passing the blended material through a
mesh screen.
[0008] In some embodiments, the dose of egg white protein per
capsule is about 0.2 mg egg white protein, 1.0 mg egg white
protein, 10.0 mg egg white protein, 20 mg or more, 100 mg or more
and the blended powder is provided in a pouch, 300 mg or more and
the blended powder is provided in a pouch, or 1000 mg or more and
the blended powder is provided in a pouch.
[0009] In other embodiments, the levels of egg white protein in the
egg white protein powder are stable in the pharmaceutical
formulation for about 3, 6, 9, 11 or more months. In some
embodiments, the concentration of one or more of ovomucoid,
ovalbumin, and lysozyme proteins in the formulation are stable for
about 3, 6, 9, or 11 or more months.
[0010] In some embodiments, the diluent is selected from the group
consisting of alginic acid and salts thereof; cellulose
derivatives; silicified microcrystalline cellulose;
microcrystalline dextrose; amylose; magnesium aluminum silicate;
polysaccharide acids; bentonites; gelatin;
polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone;
povidone; starch; pregelatinized starch; tragacanth, dextrin, a
sugar; dicalcium phosphate; a natural or synthetic gum;
polyvinylpyrrolidone, larch arabogalactan, Veegum.RTM.,
polyethylene glycol, waxes, sodium alginate, a starch; a
cross-linked starch; a cross-linked polymer; a cross-linked
polyvinylpyrrolidone; alginate; a clay; a gum; sodium starch
glycolate; bentonite; a natural sponge; a surfactant; a resin;
citrus pulp; sodium lauryl sulfate; and sodium lauryl sulfate in
combination starch. In some embodiments, the diluent is the diluent
is Prosolv, such as a mixture of Prosolv SMCC and Prosolv HD90.
[0011] In some embodiments, the filling agent is selected from the
group consisting of lactose, calcium carbonate, calcium phosphate,
dibasic calcium phosphate, calcium sulfate, microcrystalline
cellulose, cellulose powder, dextrose, dextrates, dextran,
starches, pregelatinized starch, sucrose, xylitol, lactitol,
mannitol, sorbitol, sodium chloride, and polyethylene glycol. In
some embodiments, the filling agent is mannitol.
[0012] In some embodiments, the lubricant is selected from the
group consisting of stearic acid, calcium hydroxide, talc, corn
starch, sodium stearyl fumerate, alkali-metal and alkaline earth
metal salts, stearic acid, sodium stearates, magnesium stearate,
zinc stearate, waxes, Stearowet.RTM., boric acid, sodium benzoate,
sodium acetate, sodium chloride, leucine, a polyethylene glycol or
a methoxypolyethylene glycol, propylene glycol, sodium oleate,
glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, and
magnesium or sodium lauryl sulfate. In some embodiments, the
lubricant is magnesium stearate. In some embodiments, the glidant
is talc.
[0013] Other embodiments are a method of making a capsule
formulation, comprising, (a) mixing egg white protein powder and a
diluent in a first blend; (b) discharging the blended material; (c)
passing the blended material through a mesh screen and blending the
screened material in a second blend; (d) adding in a filling agent,
glidant and/or diluent in a third blend; and (e) encapsulating the
blended powder. In some embodiments, the method also comprises
sampling the blended material of step (d) one or more times prior
to encapsulation. In some embodiments, the mesh screen of step (c)
comprises a # 20 mesh screen.
[0014] In some embodiments, the dose comprises from about 0.2 to
about 1000 mg egg white protein. In some embodiments, the egg white
protein comprises lysozyme, ovomucoid, and ovalbumin. In other
embodiments, the concentration of lysozyme, ovomucoid and ovalbumin
is characterized by Size exclusion chromatography (SEC)-HPLC. In
some embodiments, the concentration is at least an amount of a
reference standard.
[0015] In some embodiments, the encapsulated formulation is stable
for at least about 3, 6, 9, 12, 24, or 36 months. In other
embodiments, the encapsulated formulation is stable at a
temperature from about 2.degree. C. to about 30.degree. C. In some
embodiments, the capsule comprises Hydroxypropyl Methyl Cellulose
(HPMC). The method can further comprise storing the formulation in
a container, such as a bottle and the bottle can be a Hi-Density
Propyl Ethylene bottle. In some embodiments, the container can
further comprise a desiccant packet to control moisture content of
the container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a dynamic vapor sorption (DVS) Isotherm Plot
showing the rate of change in mass of a sample (rate of moisture
uptake) in accordance with aspects of the present technology.
Organic and water sorption isotherms are measured for egg white
powder.
[0017] FIG. 2 is a table showing a DVS Isotherm analysis of rate of
moisture uptake in egg white protein in accordance with aspects of
the present technology.
[0018] FIG. 3 is a plot showing DVS change in mass for egg white
protein for analyzing a rate of moisture uptake in accordance with
aspects of the present technology.
[0019] FIG. 4 is a chromatogram overlay of egg white
protein(.about.1 mg/mL protein) exposed to 1 N HCl overnight,
untreated egg white protein(.about.1 mg/mL protein stored @
4.degree. C.) and acid control sample in accordance with aspects of
the present technology.
[0020] FIG. 5 is a chromatogram overlay of egg white
protein(.about.1 mg/mL protein) exposed to 1 N NaOH overnight,
untreated egg white protein(.about.1 mg/mL protein stored @
4.degree. C.) and base control sample in accordance with aspects of
the present technology.
[0021] FIG. 6 is a chromatogram overlay of egg white
protein(.about.1 mg/mL protein) exposed to 3% hydrogen peroxide
overnight, untreated egg white protein(.about.1 mg/mL protein
stored @ 4.degree. C.) and base control sample in accordance with
aspects of the present technology.
[0022] FIG. 7 is a chromatogram overlay of egg white
protein(.about.1 mg/mL protein) exposed to 70.degree. C. heat
overnight, untreated egg white protein(.about.1 mg/mL protein
stored @ 4.degree. C.) and heat control sample in accordance with
aspects of the present technology.
[0023] FIG. 8 is a chromatogram overlay of egg white
protein(.about.1 mg/mL protein) exposed to light overnight,
untreated egg white protein(.about.1 mg/mL protein stored @
4.degree. C.) and light control sample in accordance with aspects
of the present technology.
[0024] FIG. 9 is an Enzyme-linked immunosorbent assay (ELISA) plot
showing primary antibody concentration versus absorbance at varying
dilutions of coating antigen for ovomucoid protein standard in
accordance with aspects of the present technology.
[0025] FIG. 10 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for ovomucoid protein sample in accordance with additional
aspects of the present technology
[0026] FIG. 11 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for Ovalbumin protein standard in accordance with aspects
of the present technology.
[0027] FIG. 12 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for Ovalbumin protein standard, with no secondary antibody
and in accordance with additional aspects of the present
technology.
[0028] FIG. 13 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for Ovalbumin protein sample in accordance with further
aspects of the present technology
[0029] FIG. 14 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for Lysozyme protein standard in accordance with aspects of
the present technology.
[0030] FIG. 15 is an ELISA plot showing primary antibody
concentration versus absorbance at varying dilutions of coating
antigen for Lysozyme protein sample in accordance with aspects of
the present technology.
[0031] FIG. 16 is a chromatogram overlay of (from top to bottom)
Chicken Egg Albumin (.about.0.04 mg/mL), Trypsin Inhibitor
(.about.0.04 mg/mL), Lysozyme (.about.0.04 mg/mL), and Egg White
Protein (.about.0.01 mg/ml) using two Phenomenex Yana 2000 SEC
columns in series and in accordance with aspects of the present
technology.
[0032] FIG. 17 is a chromatogram overlay of Egg White Protein
Placebo (Starch 1500), Egg White Protein Standard (.about.0.02
mg/mL protein), Placebo spiked with Egg White Protein (.about.0.02
mg/mL protein) and Egg White Protein Standard prepared with Placebo
supernatant and in accordance with aspects of the present
technology.
[0033] FIG. 18 is a chromatogram overlay of 1 mg/mL Egg White
Protein diluted with PBS solutions using size exclusion
chromatography in accordance with aspects of the present
technology.
[0034] FIG. 19 is a chromatogram overlays of 1 mg/mL Egg White
Protein diluted with 50 mM Phosphate Buffer solutions using size
exclusion chromatography in accordance with aspects of the present
technology.
[0035] FIG. 20 is a chromatogram overlay of 1 mg/mL Egg White
Protein diluted with 200 mM Phosphate Buffer solutions using size
exclusion chromatography in accordance with aspects of the present
technology.
[0036] FIG. 21 is a plot showing linearity between the sample
weight (x-axis) and nitrogen peak area (y axis) of Egg Protein
Blends in accordance with aspects of the present technology.
[0037] FIG. 22 is a chromatogram overlay of Egg White Protein
Standard (bottom) and 0.2 mg Capsule Content Uniformity Sample, Lot
#: 14009A-1 (top).
[0038] FIG. 23 is a chromatogram overlay of Egg White Protein
Standard (bottom) and 1.0 mg Blend Uniformity Sample, Lot #:
14010B-1 (top) in accordance with aspects of the present
technology.
[0039] FIG. 24 is a chromatogram overlay of Egg White Protein
Standard (bottom) and 10 mg Capsule Content Uniformity Sample, Lot
#: 14011C-1 (top) in accordance with aspects of the present
technology.
DETAILED DESCRIPTION
I. Overview
[0040] Food allergies are caused, in most cases, by a reaction to
proteins in the food. In the early years of life the immune system
is still developing and may fail to develop tolerance to dietary
antigens (this may also be described as insufficient induction of
oral tolerance). The result is that the baby or child or young
animal mounts an exaggerated immune response to the dietary protein
and develops an allergic response to it. The most common food
allergies in children are milk, eggs, peanuts, and tree nuts.
Currently there are no effective treatments available for food
allergy. Avoiding the offending allergen has been the only accepted
strategy to manage food allergy. However, strict avoidance diets
can be complicated due to difficulty in interpreting labels and by
the presence of undeclared or hidden allergens in commercially
prepared foods.
[0041] Symptoms experienced by subjects with allergy to egg
proteins can be physiologically diverse as well as have varying
severity. For example, symptoms may involve skin (atopic
dermatitis, hives/angioedema, rashes), gastrointestinal tract
(growth failure, severe gastro-esophageal reflux, chronic diarrhea,
persistent constipation, malabsorption syndromes, recurrent
vomiting, enterocolitis, anoproctite) or potentially
life-threatening anaphylactic reactions (glottis edema, hypotension
up to shock, tight asthma, acute skin and gastrointestinal
symptoms). In most cases histopathological lesions of the
intestinal mucosa are found that are very similar to the typical
ones of celiac disease (intestinal villous atrophy of various
degrees), anatomo-pathologic index of the malabsorption condition.
The intensity and the number of symptoms is variable over time, not
only from subject to subject, but also in the individual
patient.
[0042] Because individuals severely allergic to eggs can have
life-threatening reactions after consuming small amounts of egg
proteins, their quality of life can be severely impacted by their
allergy. Despite the need for treatments, clinical development of
oral immunotherapy for food allergy has proceeded slowly and no
FDA-approved oral immunotherapy treatments currently exist. The use
of food/processed food products in immunotherapy is not ideal as
the allergen levels present in foods may be inconsistent; may
degrade over time and under certain conditions; and in the case of
powdered food products, may clump or adhere to capsules or other
packaging. Because tiny amounts of food allergens can cause severe
allergic reaction, fluctuations in active ingredient for any of
these reasons could render the oral immunotherapy treatment
unpredictable.
[0043] Chemical stability is a critical aspect in the design and
manufacture, as well as regulatory review and approval, of
pharmaceutical compositions. The rate of decomposition of these
compositions may be affected by numerous environmental factors,
including temperature, light, radiation, enzyme or other catalysts,
pH and ionic strength of the solution, solvent type, and buffer
species. Such degradation may decrease efficacy and shorten
effective shelf life.
[0044] The egg protein formulations provided herein may provide for
increased shelf life and stability and may lessen the risks
associated with oral immunotherapy treatment of egg allergy by
providing for consistent dosing of egg allergens. Consistent dosing
is achieved both through the detailed characterization of the
protein levels present in the egg protein formulations (and
selection of lots meeting defined criteria), and through the
improvement in stability of the egg allergens present in such
compositions.
[0045] Specific immunotherapy for food allergy, including egg
allergy, in the forms of oral immunotherapy (OIT) and sublingual
immunotherapy (SLIT) has been studied in recent years and has
demonstrated encouraging safety and efficacy results in early
clinical trials, including beneficial immunologic changes. OIT has
shown evidence for inducing desensitization in most subjects with
immunologic changes over time indicating progression toward
clinical tolerance (Skripak et. al., J. Allergy Clin Immunol.
122(6): 1154-1160, 2008; Keet et. al., J. Allergy Clin Immunol.
129(2): 448-455, 2012).
[0046] Various aspects of the present technology provide
formulations comprising egg white protein protein that may be
formulated into a pharmaceutical composition. These presently
disclosed formulations, when administered to a patient according to
a treatment regimen, can provide oral immunotherapy (OIT) for
subjects that are allergic to eggs and egg products. Following
treatment, subjects administered an oral food challenge (OFC) may
be partially or fully desensitized to egg protein in accordance
with aspects of the present technology.
[0047] Provided herein are compositions (i.e., formulations) and
methods for oral immunotherapy of egg and egg protein products in
accordance with aspects of the present technology.
[0048] Various aspects of the present technology provide egg
protein protein formulations, methods of manufacturing egg protein
protein formulations and uses thereof. In some embodiments, an egg
protein composition can comprise one or more glidants, one or more
lubricants, and one or more diluents and/or filling agents. For
example, in other embodiments, an egg protein composition can
comprise one or more glidants, one or more lubricants, and one or
more diluents and one or more filling agents.
[0049] Specific details of several embodiments of the technology
are described below in the Detailed Description and the Examples.
Although many of the embodiments are described below with respect
to compositions (i.e., formulations) for oral immunotherapy and/or
for use in clinical trials for oral immunotherapy of egg protein,
other applications and other embodiments in addition to those
described herein are within the scope of the technology.
Additionally, several other embodiments of the technology can have
different components or procedures than those described herein. A
person of ordinary skill in the art, therefore, will accordingly
understand that the technology can have other embodiments with
additional components, or the technology can have other embodiments
without several of the aspects shown and described below.
[0050] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
II. Definitions
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the present technology described
herein belong. All patents and publications referred to herein are
incorporated by reference.
[0052] The use of individual numerical values are stated as
approximations as though the values were preceded by the word
"about" or "approximately." Similarly, the numerical values in the
various ranges specified in this application, unless expressly
indicated otherwise, are stated as approximations as though the
minimum and maximum values within the stated ranges were both
preceded by the word "about" or "approximately." In this manner,
variations above and below the stated ranges can be used to achieve
substantially the same results as values within the ranges. As used
herein, the terms "about" and "approximately" when referring to a
numerical value shall have their plain and ordinary meanings to a
person of ordinary skill in the art to which the particular subject
matter is most closely related or the art relevant to the range or
element at issue. The amount of broadening from the strict
numerical boundary depends upon many factors. For example, some of
the factors which may be considered include the criticality of the
element and/or the effect a given amount of variation will have on
the performance of the claimed subject matter, as well as other
considerations known to those of skill in the art. As used herein,
the use of differing amounts of significant digits for different
numerical values is not meant to limit how the use of the words
"about" or "approximately" will serve to broaden a particular
numerical value. Thus, as a general matter, "about" or
"approximately" broaden the numerical value. Also, the disclosure
of ranges is intended as a continuous range including every value
between the minimum and maximum values plus the broadening of the
range afforded by the use of the term "about" or "approximately."
Thus, recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it there individually recited herein.
[0053] The term "about" is used synonymously with the term
"approximately." As one of ordinary skill in the art would
understand, the exact boundary of "about" will depend on the
component of the composition. Illustratively, the use of the term
"about" indicates that values slightly outside the cited values,
i.e., plus or minus 0.1% to 10%, which are also effective and safe.
In other embodiments, the use of the term "about" indicates values
slightly outside the cited values, i.e., plus or minus 0.1% to 5%,
which are also effective and safe. In other embodiments, the use of
the term "about" indicates values slightly outside the cited
values, i.e., plus or minus 0.1% to 2%, which are also effective
and safe.
[0054] It is to be understood that any ranges, ratios and ranges of
ratios that can be formed by, or use of the terms "a" and "an" and
"the" and similar referents in the context of this disclosure
(especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., such as, preferred, preferably) provided herein, is intended
merely to further illustrate the content of the disclosure and does
not pose a limitation on the scope of the technology or the claims.
No language in the specification should be construed as indicating
any non-claimed element as essential to the present technology.
[0055] The term "absorption" typically refers to the process of
movement of egg allergen(s) from the gastrointestinal tract into a
blood vessel.
[0056] The term "animal", as used herein, refers to humans as well
as non-human animals, including, for example, mammals, birds,
reptiles, amphibians, and fish. In some embodiments, the non-human
animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a
monkey, a dog, a cat, a primate, or a pig). An animal may be a
transgenic animal.
[0057] The term "antigen", as used herein, refers to a molecule
that elicits production of an antibody response (i.e., a humoral
response) and/or an antigen-specific reaction with T-cells (i.e., a
cellular response) in an animal.
[0058] The term "allergen", as used herein, refers to a subset of
antigens which elicit the production of IgE in addition to other
isotypes of antibodies. The terms "allergen", "natural allergen",
and "wild-type allergen" may be used interchangeably. Some examples
of allergens for the purpose of the present technology are protein
allergens.
[0059] The phrase "allergic reaction", as used herein, relates to
an immune response that is IgE mediated with clinical symptoms
primarily involving the cutaneous (e.g., uticana, angiodema,
pruritus), respiratory (e.g., wheezing, coughing, laryngeal edema,
rhinorrhea, watery/itching eyes), gastrointestinal (e.g., vomiting,
abdominal pain, diarrhea), and cardiovascular (i.e., if a systemic
reaction occurs) systems. For the purposes of the present
technology, an asthmatic reaction is considered to be a form of
allergic reaction.
[0060] The phrase "anaphylactic allergen", as used herein, refers
to a subset of allergens that are recognized to present a risk of
anaphylactic reaction in allergic individuals when encountered in
its natural state, under natural conditions. For example, as
described herein pollen allergens, mite allergens, allergens in
animal danders or excretions (e.g., saliva, urine), and fungi
allergens are not considered to be anaphylactic allergens. On the
other hand, food allergens, insect allergens, and rubber allergens
(e.g., from latex) are generally considered to be anaphylactic
allergens. Food allergens, in particular, are anaphylactic
allergens for use in the practice of the present technology. In
particular, nut allergens (e.g., from egg, walnut, almond, pecan,
cashew, hazelnut, pistachio, pine nut, brazil nut), dairy allergens
(e.g., from egg, milk), seed allergens (e.g., from sesame, poppy,
mustard), soybean, wheat, and fish allergens (e.g., from shrimp,
crab, lobster, clams, mussels, oysters, scallops, crayfish) are
anaphylactic food allergens according to the present technology.
Particularly interesting anaphylactic allergens are those to which
reactions are commonly so severe as to create a risk of death.
[0061] The phrase "anaphylaxis" or "anaphylactic reaction", as used
herein, refers to a subset of allergic reactions characterized by
mast cell degranulation secondary to cross-linking of the
high-affinity IgE receptor on mast cells and basophils induced by
an anaphylactic allergen with subsequent mediator release and the
production of severe systemic pathological responses in target
organs, e.g., airway, skin, digestive tract, and cardiovascular
system. As is known in the art, the severity of an anaphylactic
reaction may be monitored, for example, by assaying cutaneous
reactions, puffiness around the eyes and mouth, vomiting, and/or
diarrhea, followed by respiratory reactions such as wheezing and
labored respiration. The most severe anaphylactic reactions can
result in loss of consciousness and/or death.
[0062] The phrase "antigen presenting cell" or "APC", as used
herein, refers to cells which process and present antigens to
T-cells to elicit an antigen-specific response, e.g., macrophages
and dendritic cells.
[0063] When two entities are "associated with" one another as
described herein, they are linked by a direct or indirect covalent
or non-covalent interaction. Preferably, the association is
covalent. Desirable non-covalent interactions include, for example,
hydrogen bonding, van der Walls interactions, hydrophobic
interactions, magnetic interactions, etc.
[0064] "Bioavailability" refers to the percentage of the weight of
egg allergen(s) dosed that is delivered into the general
circulation of the animal or human being studied. The total
exposure (AUC(0-.infin.)) of a drug when administered intravenously
is usually defined as 100% Bioavailable (F %). "Oral
bioavailability" refers to the extent to which egg allergen(s) are
absorbed into the general circulation when the pharmaceutical
composition is taken orally as compared to intravenous
injection.
[0065] "Blood plasma concentration" refers to the concentration of
an egg allergen(s) in the plasma component of blood of a subject.
It is understood that the plasma concentration of egg allergen(s)
may vary significantly between subjects, due to variability with
respect to metabolism and/or possible interactions with other
therapeutic agents. In accordance with one aspect of the present
technology, the blood plasma concentration of egg allergen(s) may
vary from subject to subject. Likewise, values such as maximum
plasma concentration (Cmax) or time to reach maximum plasma
concentration (Tmax), or total area under the plasma concentration
time curve (AUC(0-.infin.)) may vary from subject to subject. Due
to this variability, the amount necessary to constitute "a
therapeutically effective amount" of egg allergen(s) may vary from
subject to subject.
[0066] "Carrier materials" include any commonly used excipients in
pharmaceutics and should be selected on the basis of compatibility
with egg allergen(s) and the release profile properties of the
desired dosage form. Particular examples of carrier materials can
include binders, suspending agents, disintegration agents, filling
agents, surfactants, solubilizers, stabilizers, lubricants, wetting
agents, diluents, and the like.
[0067] "Excipients," as used herein are substances that can
facilitate drug delivery, absorption or solubility. Excipients can
include diluents, filling agents, lubricants, and glidants.
[0068] "Pharmaceutically compatible carrier materials" may
comprise, but are not limited to, acacia, gelatin, colloidal
silicon dioxide, calcium glycerophosphate, calcium lactate,
maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone
(PVP), cholesterol, cholesterol esters, sodium caseinate, soy
lecithin, taurocholic acid, phosphotidylcholine, sodium chloride,
tricalcium phosphate, dipotassium phosphate, cellulose and
cellulose conjugates, sugars sodium stearoyl lactylate,
carrageenan, monoglyceride, diglyceride, pregelatinized starch, and
the like. See, e.g., Remington: The Science and Practice of
Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company,
1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and
Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New
York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery
Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).
[0069] As used herein, the terms "comprising," "including," and
"such as" are used in their open, non-limiting sense.
[0070] The phrase "decreased anaphylactic reaction", as used
herein, relates to a decrease in clinical symptoms following
treatment of symptoms associated with exposure to an anaphylactic
allergen, which can involve exposure via cutaneous, respiratory,
gastrointestinal, and mucosal (e.g., ocular, nasal, and aural)
surfaces or a subcutaneous injection (e.g., via a bee sting).
[0071] "Desensitization" or "desensitize" refers to the ability of
a patient to consume small to large amounts of the allergic food
source without demonstrating an allergic reaction. Desensitization
differs from "tolerance" in that it requires chronic treatment with
the food source to maintain the "allergic-free" state. Whereas in
the "tolerance" state, treatment is no longer required.
[0072] "Diluents" are inert agents typically used for bulking or
dilution that do not have pharmacologic activity. Diluents can be
added to a small mass. Diluents for use in the formulations
provided herein include, but are not limited to, alginic acid and
salts thereof; cellulose derivatives such as
carboxymethylcellulose, methylcellulose (e.g., Methocel.RTM.),
hydroxypropylmethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose (e.g., Klucel.RTM.), ethylcellulose (e.g.,
Ethocel.RTM.), microcrystalline cellulose (e.g., Avicel.RTM.);
silicified microcrystalline cellulose (e.g., Prosolv SMCC 50.RTM.,
Prosolv HD 90.RTM.); microcrystalline dextrose; amylose; magnesium
aluminum silicate; polysaccharide acids; bentonites; gelatin;
polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone;
povidone; starch; pregelatinized starch; tragacanth, dextrin, a
sugar, such as sucrose (e.g., Dipac.RTM.), glucose, dextrose,
molasses, sorbitol, xylitol (e.g., Xylitab.RTM.), lactose (e.g.,
lactose monohydrate, lactose anhydrous, etc.); dicalcium phosphate;
a natural or synthetic gum such as acacia, tragacanth, ghatti gum,
mucilage of isapol husks, polyvinylpyrrolidone (e.g.,
Polyvidone.RTM. CL, Kollidon.RTM. CL, Polyplasdone.RTM. XL-10),
larch arabogalactan, Veegum.RTM., polyethylene glycol, waxes,
sodium alginate, a starch, e.g., a natural starch such as corn
starch or potato starch, a pregelatinized starch such as Colorcon
(Starch 1500), National 1551 or Amijel.RTM., or sodium starch
glycolate such as Promogel.RTM. or Explotab.RTM.; a cross-linked
starch such as sodium starch glycolate; a cross-linked polymer such
as crospovidone; a cross-linked polyvinylpyrrolidone; alginate such
as alginic acid or a salt of alginic acid such as sodium alginate;
a clay such as Veegum.RTM. HV (magnesium aluminum silicate); a gum
such as agar, guar, locust bean, Karaya, pectin, or tragacanth;
sodium starch glycolate; bentonite; a natural sponge; a surfactant;
a resin such as a cation-exchange resin; citrus pulp; sodium lauryl
sulfate; sodium lauryl sulfate in combination starch; and
combinations thereof. In some embodiments, the formulation
comprises microcrystalline cellulose or starch 1500. In other
embodiments, the formulation comprises microcrystalline cellulose
and starch 1500.
[0073] The term "epitope", as used herein, refers to a binding site
including an amino acid motif of between approximately six and
fifteen amino acids which can be bound by an immunoglobulin (e.g.,
IgE, IgG, etc.) or recognized by a T-cell receptor when presented
by an APC in conjunction with the major histocompatibility complex
(MHC). A linear epitope is one where the amino acids are recognized
in the context of a simple linear sequence. A conformational
epitope is one where the amino acids are recognized in the context
of a particular three dimensional structure.
[0074] "Filling agents," as used herein refers to bulking agents.
For example, inert substances that can be put into a capsule.
Filling agents for use in the formulations provided herein include,
but are not limited to, compounds such as lactose, calcium
carbonate, calcium phosphate, dibasic calcium phosphate, calcium
sulfate, microcrystalline cellulose, cellulose powder, dextrose,
dextrates, dextran, starches, pregelatinized starch, sucrose,
xylitol, lactitol, mannitol, sorbitol, sodium chloride,
polyethylene glycol, and combinations thereof.
[0075] An allergen "fragment" according to the present technology
is any part or portion of the allergen that is smaller than the
intact natural allergen. In certain embodiments of the present
technology, the allergen is a protein and the fragment is a
peptide.
[0076] "Glidants" are anti-caking agents and act to enhance the
flow of a granular mixture by reducing interparticle friction used
in the pharmaceutical production of, for example, capsules.
Glidants for use in the formulations provided herein include, but
are not limited to, colloidal silicon dioxide (Cab-O-Sil) and talc
(e.g., Ultra Talc 4000). In some embodiments, the composition
comprises talc.
[0077] The phrase "immunodominant epitope", as used herein, refers
to an epitope which is bound by antibody in a large percentage of
the sensitized population or where the titer of the antibody is
high, relative to the percentage or titer of antibody reaction to
other epitopes present in the same antigen. In some embodiments, an
immunodominant epitope is bound by antibody in more than 50% of the
sensitive population and, in further examples more than 60%, 70%,
80%, 90%, 95%, or 99%.
[0078] The phrase "immunostimulatory sequences" or "ISS", as used
herein, relates to oligodeoxynucleotides of bacterial, viral, or
invertebrate origin that are taken-up by APCs and activate them to
express certain membrane receptors (e.g., B7-1 and B7-2) and
secrete various cytokines (e.g., IL-1, IL-6, IL-12, TNF). These
oligodeoxynucleotides contain unmethylated CpG motifs and when
injected into animals in conjunction with an antigen, appear to
skew the immune response towards a Th1-type response. See, for
example, Yamamoto et al., Microbiol. Immunol. 36:983, 1992; Krieg
et al., Nature 374:546, 1995; Pisetsky, Immunity 5:303, 1996; and
Zimmerman et al., J. Immunol. 160:3627, 1998.
[0079] "Isolated" (used interchangeably with "substantially pure")
when applied to polypeptides means a polypeptide or a portion
thereof, which has been separated from other proteins with which it
naturally occurs. Typically, the polypeptide is also substantially
(i.e., from at least about 70% to about 99%) separated from
substances such as antibodies or gel matrices (polyacrylamide)
which are used to purify it.
[0080] "Lubricants," as used herein are substances that prevent
ingredients from clumping together and from sticking to the wall of
a pharmaceutical capsule or other container. Lubricants allow a
capsule to be emptied without undue loss of active ingredients.
Lubricants for use in the formulations provided herein include, but
are not limited to, stearic acid, calcium hydroxide, talc, corn
starch, sodium stearyl fumerate, alkali-metal and alkaline earth
metal salts, such as aluminum, calcium, magnesium, zinc, stearic
acid, sodium stearates, magnesium stearate, zinc stearate, waxes,
Stearowet.RTM., boric acid, sodium benzoate, sodium acetate, sodium
chloride, leucine, a polyethylene glycol or a methoxypolyethylene
glycol such as CarbowaxTM, PEG 4000, PEG 5000, PEG 6000, propylene
glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate,
glyceryl benzoate, magnesium or sodium lauryl sulfate, and
combinations thereof
[0081] A "measurable serum concentration" or "measurable plasma
concentration" describes the blood serum or blood plasma
concentration, typically measured in mg, .mu.g, or ng of
therapeutic agent per ml, dl, or l of blood serum, absorbed into
the bloodstream after administration. As used herein, measurable
plasma concentrations are typically measured in ng/ml or
.mu.g/ml.
[0082] "Oral food challenge" refers to a highly accurate diagnostic
test for food allergy. During the food challenge, the allergist
feeds the patient the suspect food in measured doses, starting with
very small amounts that are unlikely to trigger symptoms. Following
each dose, the patient is observed for a period of time for any
signs of a reaction. If there are no symptoms, the patient
gradually receives increasingly larger doses. If any signs of a
reaction are evident, the food challenge is stopped and the patient
is characterized as failing the food challenge and is allergic to
the food at the sensitivity level determined by the amount of food
triggering the allergic response.
[0083] "Oral immunotherapy" refers to an orally-administered
medical treatment for patients suffering from allergies, involving
administering increasing doses of allergens to the patients in
order to desensitize or provide tolerance to a patient for that
allergen.
[0084] "Pharmacodynamics" refers to the factors which determine the
biologic response observed relative to the concentration of drug at
a site of action.
[0085] "Pharmacokinetics" refers to the factors which determine the
attainment and maintenance of the appropriate concentration of drug
at a site of action.
[0086] "Plasticizers" are compounds which may be used to soften the
microencapsulation material or film coatings to make them less
brittle. Suitable plasticizers include, e.g., polyethylene glycols
such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,
stearic acid, propylene glycol, oleic acid, triethyl cellulose and
triacetin. In some embodiments, plasticizers can also function as
dispersing agents or wetting agents.
[0087] "Solubilizers" include compounds such as triacetin,
triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl
sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide,
N-methylpyrrolidone, N-hydroxyethylpyrrolidone,
polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl
cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol,
bile salts, polyethylene glycol 200-600, glycofurol, transcutol,
propylene glycol, dimethyl isosorbide and combinations thereof.
[0088] The total egg protein, ovomucoid protein, ovalbumin protein,
and/or lysozyme protein in the egg protein formulations provided
herein may be considered "stable" if its concentration is .+-.10%
the original concentration of such protein(s) in the egg protein
formulation immediately after manufacture.
[0089] The compositions described herein can be formulated for
administration to a subject via any conventional means including,
but not limited to, oral administration routes. As used herein, the
term "subject" is used to mean an animal, such as a mammal,
including a human or non-human. The formulations are for prevention
and treatment of symptoms associated with exposure to limited
amounts of egg allergen in children and adults. In some
embodiments, a subject is from about 1 to about 35 years of age,
including from 4 to about 26 years of age.
[0090] A "therapeutically effective amount" or "effective amount"
is that amount of egg allergen(s) needed to achieve a
pharmacological effect. The term "therapeutically effective amount"
includes, for example, a prophylactically effective amount. An
"effective amount" of egg allergen(s) is an amount effective to
achieve a desired pharmacologic effect or therapeutic improvement
without undue adverse side effects. The effective amount of an egg
allergen(s) will be selected by those skilled in the art depending
on the particular subject and the disease level. It is understood
that "an effect amount" or "a therapeutically effective amount" can
vary from subject to subject, due to variation in metabolism, age,
weight, general condition of the subject, the condition being
treated, the severity of the condition being treated, and the
judgment of the prescribing physician.
[0091] "Tolerance" to an allergen refers to the relatively
long-lasting effects of immunotherapy, presumably due to effects on
T cell responsiveness, that persist even after the treatment is
discontinued (although tolerance may not always be permanent).
[0092] "Treat" or "treatment" as used in the context of an
allergy-related disorder refers to any treatment of a disorder or
disease related to allergy, such as preventing the disorder or
disease from occurring in a subject which may be predisposed to the
disorder or disease, but has not yet been diagnosed as having the
disorder or disease; inhibiting the disorder or disease, e.g.,
arresting the development of the disorder or disease, relieving the
disorder or disease, causing regression of the disorder or disease,
relieving a condition caused by the disease or disorder, or
stopping the symptoms of the disease or disorder.
III. Compositions/Formulations
[0093] Provided herein are egg formulations and/or egg protein
formulations for use in oral immunotherapy. In some embodiments,
the formulation comprises egg protein powder, or alternatively, one
or more proteins isolated from egg powder, blended with one or more
excipients. For example, in addition to egg protein or protein(s)
isolated from egg powder, the formulations can comprise one or more
of each of diluents, filling agents, glidants, lubricants,
colorants, and capsule shell components.
[0094] In some embodiments, egg white protein comprises ovomucoid,
ovalbumin, and lysozyme proteins. In other embodiments, egg white
protein contains as active ingredients: ovomucoid, ovalbumin, and
lysozyme proteins.
[0095] In some embodiments, an egg protein formulation comprises
one or more diluents. In some embodiments, an egg protein
formulation comprises one or more glidants. In some embodiments, an
egg protein formulation comprises one or more lubricants. In some
embodiments, an egg protein formulation comprises one or more
filling agents.
[0096] In some embodiments, a final egg protein formulation
comprises egg white protein (containing characterized egg allergen
proteins ovomucoid, ovalbumin and lysozyme) formulated with a
diluent, a filling agent, and a lubricant in graduated doses,
having total egg white protein doses of 0.2 mg, 1 mg, 10 mg, 20 mg,
100 mg, 200 mg and 1000 mg each of egg white protein. Each capsule
or container formulation (e.g., sachet) may be opened and the
content mixed into taste-masking food immediately prior to
administration.
[0097] In other embodiments, a final formulation comprises egg
white protein (containing characterized egg allergen proteins
ovomucoid, ovalbumin and lysozyme) formulated with a diluent and a
filling agent in graduated doses, having total egg white protein
doses of 0.2 mg, 1.0 mg, 10 mg, 20 mg, 100 mg, 200 mg, 300 mg, 500
mg, or 1000 mg of egg white protein. Each capsule or container
(e.g., pouch) may be opened and the content mixed into
taste-masking food immediately prior to administration.
[0098] In some embodiments, the formulation comprising egg protein
is encapsulated (e.g., 0.2 mg, 1.0 mg, 10 mg, 20 mg, and 100 mg
doses). In other embodiments, the formulation comprising egg
protein is sachet packaged (e.g., 100 mg, 200 mg, 300 mg, 500 mg,
and 1000 mg doses).
[0099] The product is intended to deliver increasing concentrations
of dry egg powder, yielding egg protein at dosages of 0.2 mg, 1.0
mg, 10.0 mg, 20 mg, 100.0 mg, 200.0 mg, 500.0 mg, and 1000.0 mg.
The 0.2 mg through the 100.0 mg dosages may be encapsulated. The
300 mg, 500.0 mg and 1000 mg dosages may be sachet (pouched)
packaged. In some embodiments, a sachet can be a multi-layered
pouch lined, for example, with a pharmaceutically accepted and/or
compatible liner (e.g., foil). In conventional practice, and in
some embodiments, the sachet is machine-formed following filling of
the material with the desired amount of the pharmaceutical
composition. It is desired that the capsule and sachet pack
contents, at each dosage strength, empty as cleanly and completely
as possible from the capsule shells or sachet pack film as the
intent, in some embodiments, is to add the capsule or sachet pack
contents, as a powder, to food for consumption by the patient. A
placebo will be developed for each of the dosage strengths. Each
placebo will be encapsulated or sachet packaged to match its
corresponding active dosage.
[0100] In yet other embodiments, degradation of egg protein, as
measured by size exclusion chromatography, may be used to determine
stability. In a further embodiment, an egg protein formulation that
does not have significant changes in moisture content, appearance
and odor for over three months of storage (e.g., storage at
5.degree. C./60% relative humidity, storage at 25.degree. C./60%
relative humidity, storage at 40.degree. C./75% relative humidity)
can be determined to be stable. In some embodiments, the levels of
egg protein are stable for 3, 6, 9, 11, 12, 18, 24, or 36 or more
months. In a further embodiment, the levels of ovomucoid, ovalbumin
and lysozyme proteins are stable for 3, 6, 9, 11, 12, 18, 24, or 36
or more months.
[0101] Various sources of egg white protein are commercially
available. For example, the egg white protein can be Deb El Egg
White Protein from Deb El Food Products, Elizabeth N.J. or EWP from
Michael Foods, Minnetonka, Minn. The egg white protein may be
further processed under cGMP conditions. In other embodiments, the
egg white protein (approximately 85% egg protein w/w) comprises
particles, wherein the particles can have diameters in the range of
about 10 .mu.m to about 250 .mu.m, including 75 .mu.m and 150
.mu.m.
[0102] Under cGMP manufacturing conditions, the egg white protein
is formulated with a diluent, a filling agent, a glidant, and/or a
lubricant, and is subsequently encapsulated as 0.2, 1, 10, 20, 100,
200, 300, 500 or 1000 mg of egg protein in size 3 or 00
Hydroxypropyl Methyl Cellulose (HPMC) capsules or sachet packaged
(for higher doses). In certain embodiments, the concentration of
egg protein can be from about 0.05% to about 50% w/w, or any
integer therein. In other embodiments, a composition described
herein comprises one or more egg proteins in a concentration from
about 0.1% to about 25% w/w. In other embodiments, a composition
described herein comprises one or more egg proteins in a
concentration from about 0.2%, about 1%, about 2%, about 4%, or
about 50% w/w. In other embodiments, at higher doses, the sachets
comprise one or more egg white proteins in a concentration from
about 40% to about 100%, including, but not limited to 50%, 60%,
70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. In
other embodiments, a composition described herein comprises one or
more egg proteins in a concentration from about 0.1%, about 0.67%,
about 2.1%, about 4%, or about 21% w/w of formulation.
[0103] In some embodiments, a composition described herein
comprises one or more egg proteins in a target unit weight from
about 0.2 mg/capsule to about 1000 mg/sachet, or any integer
therein. In yet other embodiments, a composition described herein
comprises one or more egg proteins in a target unit weight of about
0.2 mg/capsule to about 1 mg/capsule, about 10 mg/capsule, about
100 mg/capsule or pouch, about 300 mg/capsule or pouch, or about
1000 mg/sachet or pouch.
[0104] Compositions for use in the methods described herein
include, but are not limited to, about 0.2 mg, about 1.0 mg, about
10 mg, about 20 mg, about 100 mg, about 200 mg, about 300 mg, about
500 mg, and/or about 1000 mg doses of total egg protein.
[0105] In some embodiments, the final formulation comprising egg
white protein is in a dose of 0.2 mg and the concentration of
ovomucoid comprises from about 0.032 to about 0.048 mg. In other
embodiments, the dose of the composition is 1.0 mg and the
concentration of ovomucoid comprises from about 0.16 to about 0.24
mg; In other embodiments, the dose of the composition is 10.0 mg
and the concentration of ovomucoid comprises from about 1.6 to
about 2.4 mg; or In other embodiments, the dose of the composition
is 100.0 mg and the concentration of ovomucoid comprises from about
16 to about 24 mg. In some embodiments, the dose of the composition
is 0.2 mg and the concentration of ovalbumin comprises from about
0.092 to about 0.108 mg; In other embodiments, the dose of the
composition is 1.0 mg and the concentration of ovalbumin comprises
from about 0.46 to about 0.54 mg; In other embodiments, the dose of
the composition is 10.0 mg and the concentration of ovalbumin
comprises from about 4.6 to about 5.4 mg; or In other embodiments,
the dose of the composition is 100.0 mg and the concentration of
ovalbumin comprises from about 46 to about 54 mg. In some
embodiments, the dose of the composition is 0.2 mg and the
concentration of lysozyme comprises from about 0.002 to about 0.018
mg; In other embodiments, the dose of the composition is 1.0 mg and
the concentration of lysozyme comprises from about 0.01 to about
0.09 mg; In other embodiments, the dose of the composition is 10.0
mg and the concentration of lysozyme comprises from about 0.1 to
about 0.9 mg; or In other embodiments, the dose of the composition
is 100.0 mg and the concentration of lysozyme comprises from about
1.0 to about 9.0 mg.
[0106] In some embodiments, a composition provided herein is
contained within a capsule including, but not limited to a white
opaque HPMC capsule shell (e.g., Capsugel) and may further contain,
in some instances, a coloring agent (e.g., pigment blends, and/or
color). In other embodiments, the capsule can be a clear or opaque
HPMC capsule shell or a blue opaque capsule shell.
[0107] In some embodiments, a composition described herein
comprises egg protein in a target unit weight from about 0.2
mg/capsule to about 1000 mg/sachet , or any integer therein. In yet
other embodiments, a composition described herein comprises egg
protein in a target unit weight of about 0.2 mg/capsule to about 1
mg/capsule, about 10 mg/capsule, about 20 mg/capsule, about 100
mg/capsule or sachet, about 200 mg/sachet, about 300 mg/sachet,
about 500 mg/sachet, or about 1000 mg/sachet.
[0108] The diluent and/or filling agent provides the opportunity to
formulate the low and high doses to contain adequate volume for
dispersal from the opened capsule. The glidants and lubricant add
flowability to the egg white protein such that the capsule can be
reproducibly filled by automated encapsulation machines, and to
optimize the efficiency by which the capsule is easily emptied of
protein by the subject. For clinical trials, the capsules can be
bulk packed into high density polyethylene (HDPE) bottles. At the
time of use, capsule(s) comprising egg protein can be opened and
the content mixed into taste-masking food immediately prior to
administration.
[0109] The concentration of diluent in an egg protein formulation
described herein may be from about 30% to about 99% w/w. In some
embodiments, the concentration of diluent may be from about 40% to
about 90% w/w of the composition. In some embodiments, the diluent
may be silicified microcrystalline cellulose and the concentration
may be about 50% to about 90% w/w of the composition. In other
embodiments, the diluent may be silicified microcrystalline
cellulose and the concentration may be about 45%, about 46%, about
47%, about 48%, about 49%, about 50%, about 51%, about 52%, about
53%, about 54%, about 55%, about 56%, about 57%, about 58%, about
59%, about 60%, about 61%, about 62%, about 63%, about 64%, about
65%, about 66%, about 67%, about 68%, about 69%, about 70%, about
71%, about 72%, about 73%, about 74%, about 75%, about 76%, about
77%, about 78%, about 79%, about 80%, about 81%, about 82%, about
83%, about 84%, about 85%, about 86%, 87, about 88%, about 89%, or
about 90% w/w of the composition. In some embodiments, more than
one type of silicified microcrystalline cellulose is used to get
the desired w/w of the composition.
[0110] The concentration of glidant in a composition described
herein may be from about 0.01% to about 10% w/w of the composition.
In some embodiments, the glidant in a composition described herein
may be about 0.01% to about 3.0%. In some embodiments, the glidant
is talc and the concentration of glidant in a composition described
herein may be about 0.01%, about 0.05%, about 0.1%, about 0.25%,
about 0.2%, about 0.75%, about 1.0%, about 1.25%, 1.5% or about
2.5% w/w of the composition.
[0111] The target unit weight of the glidant may be from about .05
to about 5 mg/capsule. In some embodiments, the glidant is talc and
the target unit weight is about 0.725, about 2.625 or about 3.0
mg/capsule. In other embodiments, the glidant is colloidal silicon
dioxide (e.g., Cab-O-Sil) and the target unit weight is about 0.5
mg, about 1.0 mg, about 2 mg, about 3.0 mg, or about 5
mg/capsule.
[0112] The concentration of lubricant in a composition described
herein may be from about 0.01% to about 10% w/w of the composition.
In some embodiments, the lubricant in a composition described
herein may be about 0.1% to about 1.0%. In some embodiments, the
lubricant is magnesium stearate and the concentration of lubricant
in a composition described herein may be about 0.01%, about 0.05%,
about 0.1%, about 0.25%, about 0.4%, about 0.5%, about 0.75%, about
1.0%, about 1.25%, or about 1.5% w/w of the composition.
[0113] The target unit weight of the lubricant may be from about
.05 to about 5 mg/capsule. In some embodiments, the lubricant is
magnesium stearate and the target unit weight is about 0.75, about
0.79 or about 2.4 mg/capsule.
[0114] The concentration of filling agent in a composition
described herein may be from about 1% to about 30% w/w of the
composition. In some embodiments, the filling agent in a
composition described herein may be about 10% to about 15%. In some
embodiments, the filling agent is mannitol and the concentration of
filling agent in a composition described herein may be about 5%,
about 10%, about 15%, or about 20% w/w of the composition.
[0115] The target unit weight of the filling agent may be from
about 15.0 to about 47.5 mg/capsule. In some embodiments, the
filling agent is mannitol and the target unit weight is about 7.5,
about 15, about 15.8, or about 47.5 mg/capsule.
[0116] It will be understood that quantitative formulas will be
adjusted depending on manufacturing final fill weights. Final fill
weights may vary from about 150 mg to about 450 mg to about 1000
mg. In some embodiments, an egg protein formulation containing
about 0.2 mg egg protein is manufactured with a final fill weight
of about 158 mg. In other embodiments, an egg protein formulation
containing about 1.0 mg egg protein is manufactured with a final
fill weight of about 150 mg. In other embodiments, an egg protein
formulation containing about 10.0 mg egg protein is manufactured
with a final fill weight of about 450 mg. In other embodiments, an
egg protein formulation containing about 100 mg egg protein is
manufactured with a final fill weight of about 450 mg.
[0117] In some embodiments, solid dosage forms may be in the form
of a tablet, (including a suspension tablet, a fast-melt tablet, a
bite-disintegration tablet, a rapid-disintegration tablet, an
effervescent tablet, or a caplet), a pill, a powder (including a
sterile packaged powder (such as a "stick pack" or foil pouch), a
dispensable powder, or an effervescent powder) a capsule (including
both soft or hard capsules, e.g., capsules made from animal-derived
gelatin or plant-derived HPMC, or "sprinkle capsules"), solid
dispersion, solid solution, pellets, or granules. In other
embodiments, the formulation is in the form of a powder.
Additionally, formulations may be administered as a single capsule
or in multiple capsule dosage form. In some embodiments, the
formulation is administered in two, or three, or four, capsules or
tablets or powder packages.
[0118] In some embodiments, solid dosage forms, e.g., tablets,
effervescent tablets, and capsules, are prepared by mixing egg
white protein comprising characterized egg allergens with one or
more pharmaceutical excipients to form a bulk blend composition.
When referring to these bulk blend compositions as homogeneous, it
is meant that the particles are dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms, such as tablets, pills, and
capsules. The individual unit dosages may also comprise film
coatings, which disintegrate upon oral ingestion or upon contact
with diluent. These formulations can be manufactured by
conventional pharmacological techniques.
[0119] Conventional pharmacological techniques include, e.g., one
or a combination of methods: (1) dry mixing, (2) direct
compression, (3) milling, (4) dry or non-aqueous granulation, (5)
wet granulation, or (6) fusion. See, e.g., Lachman et al., The
Theory and Practice of Industrial Pharmacy (1986). Other methods
include, e.g., spray drying, pan coating, melt granulation,
granulation, fluidized bed spray drying or coating (e.g., Wurster
coating), tangential coating, top spraying, tableting, extruding
and the like.
[0120] The pharmaceutical solid dosage forms described herein can
comprise the compositions described herein and one or more
pharmaceutically acceptable additives such as a compatible carrier,
binder, filling agent, suspending agent, flavoring agent,
sweetening agent, disintegrating agent, dispersing agent,
surfactant, lubricant, colorant, diluent, solubilizer, moistening
agent, plasticizer, stabilizer, penetration enhancer, wetting
agent, anti-foaming agent, antioxidant, preservative, or one or
more combination thereof. In still other aspects, using standard
coating procedures, such as those described in Remington's
Pharmaceutical Sciences, 20th Edition (2000), a film coating is
provided around the formulation. In some embodiments, some or all
of the particles are coated. In other embodiments, some or all of
the particles are microencapsulated. In yet other embodiments, some
or all of the egg allergens are amorphous material coated and/or
microencapsulated with inert excipients. In still other
embodiments, the particles not microencapsulated and are
uncoated.
[0121] Compressed tablets are solid dosage forms prepared by
compacting the bulk blend formulations described above. In various
embodiments, compressed tablets which are designed to dissolve in
the mouth will comprise one or more flavoring agents. In other
embodiments, the compressed tablets will comprise a film
surrounding the final compressed tablet. In some embodiments, the
film coating can provide a delayed release of the formulation. In
other embodiments, the film coating aids in subject compliance
(e.g., Opadry.RTM. coatings or sugar coating). Film coatings
comprising Opadry.RTM. typically range from about 1% to about 3% of
the tablet weight. In other embodiments, the compressed tablets
comprise one or more excipients.
[0122] A capsule may be prepared, e.g., by placing the bulk blend
formulation, described above, inside of a capsule. In some
embodiments, the formulations (non-aqueous suspensions and
solutions) are placed in a soft gelatin capsule. In other
embodiments, the formulations are placed in standard gelatin
capsules or non-gelatin capsules such as capsules comprising HPMC.
In other embodiments, the formulations are placed in a sprinkle
capsule, wherein the capsule may be swallowed whole or the capsule
may be opened and the contents sprinkled on food prior to eating.
In some embodiments, the therapeutic dose is split into multiple
(e.g., two, three, or four) capsules. In some embodiments, the
entire dose of the formulation is delivered in a capsule form.
[0123] In various embodiments, the particles and one or more
excipients are dry blended and compressed into a mass, such as a
tablet, having a hardness sufficient to provide a pharmaceutical
composition that substantially disintegrates within less than about
30 minutes, less than about 35 minutes, less than about 40 minutes,
less than about 45 minutes, less than about 50 minutes, less than
about 55 minutes, or less than about 60 minutes, after oral
administration, thereby releasing the formulation into the
gastrointestinal fluid.
[0124] In some aspects, dosage forms may include microencapsulated
formulations. In some embodiments, one or more other compatible
materials are present in the microencapsulation material. Exemplary
materials include, but are not limited to, pH modifiers, erosion
facilitators, anti-foaming agents, antioxidants, flavoring agents,
and carrier materials such as binders, suspending agents,
disintegration agents, filling agents, surfactants, solubilizers,
stabilizers, lubricants, wetting agents, and diluents.
[0125] Materials useful for the microencapsulation described herein
include materials compatible with egg allergens which sufficiently
isolate egg allergens from other non-compatible excipients.
Materials compatible with egg allergens are those that delay the
release of the egg allergens in vivo.
[0126] Examples of microencapsulation materials useful for delaying
the release of the formulations include, but are not limited to,
hydroxypropyl cellulose ethers (HPC) such as Klucel.RTM. or Nisso
HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),
hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,
Pharmacoat.RTM., Metolose SR, Methocel.RTM.-E, Opadry YS, PrimaFlo,
Benecel MP824, and Benecel MP843, methylcellulose polymers such as
Methocel.RTM.-A, hydroxypropylmethylcellulose acetate stearate
Aqoat (HF-LS, HF-LG, HF-MS) and Metolose.RTM., Ethylcelluloses (EC)
and mixtures thereof such as E461, Ethocel.RTM., Aqualon.RTM.-EC,
Surelease.RTM., Polyvinyl alcohol (PVA) such as Opadry AMB,
hydroxyethylcelluloses such as Natrosol.RTM.,
carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC)
such as Aqualon.RTM.-CMC, polyvinyl alcohol and polyethylene glycol
co-polymers such as Kollicoat IR.RTM., monoglycerides (Myverol),
triglycerides (KLX), polyethylene glycols, modified food starch,
acrylic polymers and mixtures of acrylic polymers with cellulose
ethers such as Eudragit.RTM. EPO, Eudragit.RTM. L30D-55,
Eudragit.RTM. FS 30D Eudragit.RTM. L100-55, Eudragit.RTM. L100,
Eudragit.RTM. 5100, Eudragit.RTM. RD100, Eudragit.RTM. E100,
Eudragit.RTM. L12.5, Eudragit.RTM. 512.5, Eudragit.RTM. NE30D, and
Eudragit.RTM. NE 40D, cellulose acetate phthalate, sepifilms such
as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures
of these materials.
[0127] Microencapsulated egg allergens may be formulated by methods
known by one of ordinary skill in the art. Such known methods
include, e.g., spray drying processes, spinning disk-solvent
processes, hot melt processes, spray chilling methods, fluidized
bed, electrostatic deposition, centrifugal extrusion, rotational
suspension separation, polymerization at liquid-gas or solid-gas
interface, pressure extrusion, or spraying solvent extraction bath.
In addition to these, several chemical techniques, e.g., complex
coacervation, solvent evaporation, polymer-polymer incompatibility,
interfacial polymerization in liquid media, in situ polymerization,
in-liquid drying, and desolvation in liquid media could also be
used. Furthermore, other methods such as roller compaction,
extrusion/spheronization, coacervation, or nanoparticle coating may
also be used.
[0128] The formulations described herein are administered and dosed
in accordance with good medical practice, taking into account the
clinical condition of the individual subject, the site and method
of administration, scheduling of administration, and other factors
known to medical practitioners.
IV. Methods of Use
[0129] The formulations described herein may be used in oral
immunotherapy (OIT) to treat a subject suffering from an egg
allergy.
[0130] Eggs and egg white protein powder are common foods and
additives found in many food products. The present egg protein
formulations may include relatively small quantities (0.2 to 100
mg/capsule) of egg proteins compared to the quantities contained in
many food products and may be delivered via the same route as
orally ingested egg-containing products.
[0131] A subject treated with the formulations described herein may
exhibit a decreased anaphylactic reaction, relating to a decrease
in clinical symptoms following treatment of symptoms associated
with exposure to an anaphylactic allergen, which can involve
exposure via cutaneous, respiratory, gastrointestinal, and mucosal
(e.g., ocular, nasal, and aural) surfaces or a subcutaneous
injection (e.g., via a bee sting) following treatment. In some
embodiments, a subject may exhibit a decreased anaphylactic
reaction of about 2%, about 5%, about 10%, about 15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about 90% or more compared to a subject receiving a
placebo or a subject not receiving treatment.
[0132] A subject treated with a composition described herein may
exhibit a decreased humoral response and/or T cell response
following treatment. In some embodiments, a subject may exhibit a
decreased humoral response and/or T cell response of about 2%,
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or
more compared to a subject receiving a placebo or a subject not
receiving treatment.
[0133] A subject treated with a composition described herein may
exhibit a decreased IgE response and/or a decreased mast cell
response following treatment. In some embodiments, a subject may
exhibit a decreased IgE response and/or a decreased mast cell
response of about 2%, about 5%, about 10%, about 15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about 90% or more compared to a subject receiving a
placebo or a subject not receiving treatment.
[0134] A subject treated with the formulation may also exhibit an
increased IgG4 response which replaces the IgE antibodies and
tempers the immune response to allergens thus lessening the
likelihood of an allergic reaction.
[0135] A subject treated with the formulations described herein may
be better able to withstand an oral food challenge (OFC) following
treatment.
[0136] A subject treated with a composition described herein may be
desensitized to egg allergy following treatment. In some
embodiments, a subject may be desensitized by about 2%, about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90% or more
compared to a subject receiving a placebo or a subject not
receiving treatment.
[0137] The compositions described herein may be administered in an
escalation schedule. In some embodiments, escalating doses are
administered to the subject on day 1 of treatment. For example, a
subject may be administered, 1, 2, 3, 4 or 5 doses of a composition
described herein on day 1. In another example, a subject may be
administered 5 doses of a composition described herein in 30 minute
increments on day 1. Subjects return on day 2 and receive a maximum
tolerated dose. Subjects with moderate symptoms observed on day 2
may return on day 3 for the next lower dose under observation in a
monitored clinic setting. Subjects able to withstand treatment on
the initial day of treatment may be administered one or more
further doses of a composition described herein.
[0138] In some embodiments, a subject is further administered 1, 2,
3, 4, 5, 6, 7, 8 or 9 additional escalating doses of a composition
described herein. The additional escalating doses may be
administered to a subject in two-week intervals.
[0139] Following the final administration, the subject may, in some
instances, be subject to an oral food challenge to determine if the
subject has been desensitized to egg allergy.
[0140] In some embodiments, the initial day escalation schedule is
shown in Table 1.
TABLE-US-00001 TABLE 1 Initial Day Escalation Schedule Cumulative
Egg protein Egg protein Formulation Formulation Dose if no Dose #
Dose De-escalation 1 0.2 mg 0.2 mg 2 0.4 mg 0.6 mg 3 0.8 mg 1.4 mg
4 1.6 mg 3.0 mg 5 3.0 mg 6.0 mg
[0141] Doses are administered at a frequency standard of every 30
minutes. Subjects at the end of the first day, tolerating less than
1.6 mg single dose may, in some cases, be considered an initial day
escalation desensitization failure.
[0142] Subjects tolerating a 1.6 or 3 mg single dose may proceed
with the greatest tolerated dose to be given daily (first dose
given in clinic setting under observation). All escalations occur
after at least 2 weeks and single dose increases in the clinic from
1.6 to 3 mg may be attempted.
[0143] All subjects return on day 2 and receive their maximum
tolerated dose under direct observation. Subjects with moderate
symptoms observed on day 2 will return on day 3 for the next lower
dose under observation in monitored clinic setting. Doses on day 2,
3 and 4 may be at least 1.6 mg or the subject, in some instances,
may be considered an escalation failure.
[0144] Following the initial escalation, and if a subject does not
have an adverse event, the escalation dose schedule shown in Table
2 may be followed in some embodiments.
TABLE-US-00002 TABLE 2 Escalation Dose Schedule Dose # Dose
(Protein) Interval (weeks) % Increase 6 6 mg 2 7 12 mg 2 100% 8 20
mg 2 67% 9 40 mg 2 100% 10 80 mg 2 100% 11 120 mg 2 50% 12 160 mg 2
33% 13 200 mg 2 25% 14 240 mg 2 20% 15 300 mg 2 25%
[0145] In other embodiments, an escalation dosing schedule can
include day 1 escalation to 50 mg and a 32-week dose escalation
with daily dosing as shown in Table 3.
TABLE-US-00003 TABLE 3 Initial Day and Escalation Dosing Schedule
Dose # Dose (Protein) Interval (weeks) % Increase 1-10 50 mg
Escalation to 50 mg dose 11 80 mg 2 60% 12 100 mg 2 25% 13 120 mg 2
20% 14 160 mg 2 33% 15 200 mg 2 25% 16 250 mg 2 25% 17 300 mg 2 30%
18 360 mg 2 20% 19 440 mg 2 22% 20 500 mg 2 14% 21 700 mg 2 40% 22
1000 mg 2 43% 23 1200 mg 2 20% 24 1500 mg 2 25% 25 1800 mg 2 20% 26
2000 mg 2 11%
[0146] In some embodiments of such methods, immediately prior to
administration, an encapsulated capsule formulation may be broken
apart and the ingredients mixed into taste -masking food.
[0147] In other embodiments, subjects continue taking active
treatment for a 3-, 6-, 12-, 24-month or longer maintenance period.
In other embodiment, subjects are updosed to 300 mg (as per dose
17) and then they are maintained at 300 mg for a long period of
time (minimum 3-6 months). In other embodiments, the subjects are
updosed to 1000 mg and then maintained at 1000 mg for a long period
of time (minimum 3-6 months) and up to years or a lifetime. These
differ from continual updosing in that the subject is updosed to a
specific dose and maintained for a long period of time.
[0148] Subjects may be monitored for onset of systemic symptoms
including, for example, flushing, intensive itching on the skin,
and sneezing and runny nose. Sense of heat, general discomfort and
agitation/anxiety may also occur.
[0149] In some embodiments, the formulations provided herein are
administered one or more days to a subject suffering from an egg
allergy.
[0150] In some embodiments, the subject is able to increase the
amount of protein they can consume without an allergic reaction by
at least about 100% compared to a subject administered a placebo or
not receiving treatment.
[0151] In other embodiments, the subject exhibits a reduced humoral
response and/or a reduced T cell response. In other embodiments,
the subject exhibits reduced anaphylaxis, a reduced mast cell
response, a reduced IgE response, reduced hives, or a combination
thereof
[0152] In some embodiments, a formulation provided herein may be
administered in conjunction with a food product.
[0153] A subject may be administered 1, 2, 3, 4 or 5 doses of a
formulation provided herein on the first day of treatment. In some
embodiments, a subject is administered 10 doses on the first day of
treatment. In other embodiments, the subject is administered said
doses in 30 minute intervals. The method may, in some instances
further comprise one or more additional treatments. In some
embodiments, the one or more additional treatments comprise
administration of a composition in two-week intervals. In other
embodiments, the one or more additional treatments comprise 1, 2,
3, 4, 5, 6, 7, 8, 9, or more doses of a composition.
[0154] Provided herein is a method of desensitizing a subject
suffering from an egg allergy comprising administering one or more
doses of a composition in accordance with aspects of the present
technology, wherein the method can comprise the following steps:
(a) administering to the subject escalating doses of 0.2 mg, 1.0
mg, 1.5 mg. 3.0 mg, and 6.0 mg in 30-minute intervals on day 1 of
the treatment regimen; (b) optionally, administering to the patient
a maximum tolerated dose on day 2 of the treatment regimen; and (c)
administering to the subject single doses of 12 mg, 20 mg, 40 mg,
80 mg, 120 mg, 160 mg, 200 mg, 240 mg, 300 mg, 360 mg, 440 mg, 500
mg, 700 mg, 1000 mg, 1200 mg, 1500 mg, 1800 mg, and 2000 mg in
two-week intervals.
[0155] In some embodiments, the method can further comprise
administering an oral food challenge (OFC) following completion of
the treatment regimen.
V. Combination Therapies
[0156] The formulations and methods described herein may also be
used in conjunction with other well-known therapeutic compounds
that are selected for their particular usefulness against the
condition that is being treated. In general, the formulations
described herein and, in embodiments where combinational therapy is
employed, other compounds, do not have to be administered in the
same formulation, and may, because of different physical and
chemical characteristics, have to be administered by different
routes, or they may be combined in a single formulation. The
determination of the mode of administration and the advisability of
administration, where possible, in the same formulation, is well
within the knowledge of the skilled clinician. The initial
administration can be made according to established protocols known
in the art, and then, based upon the observed effects, the dosage,
modes of administration and times of administration can be modified
by the skilled clinician.
[0157] The particular choice of compounds used will depend upon the
diagnosis of the attending physicians and their judgment of the
condition of the subject and the appropriate treatment protocol.
The compounds may be administered concurrently (e.g.,
simultaneously, essentially simultaneously or within the same
treatment protocol) or sequentially, depending upon the condition
of the subject, and the actual choice of compounds used. The
determination of the order of administration, and the number of
repetitions of administration of each therapeutic agent during a
treatment protocol, is well within the knowledge of the skilled
physician after evaluation of the severity of egg allergy being
treated and the condition of the subject.
[0158] It is understood that the dosage regimen to treat, prevent,
or ameliorate egg allergy, can be modified in accordance with a
variety of factors. These factors include the age, weight, sex,
diet, and/or medical condition of the subject. Thus, the dosage
regimen actually employed can vary widely and therefore can deviate
from the dosage regimens set forth herein.
[0159] The time period between the multiple administration steps
may range from, a few minutes to several hours, depending upon the
properties of each pharmaceutical agent, such as potency,
solubility, bioavailability, plasma half-life and kinetic profile
of the pharmaceutical agent. Circadian variation of the target
molecule concentration may also determine the optimal dose
interval.
[0160] In some embodiments, the formulation is administered with at
least one other anti-histamine agent, corticosteroid, beta agonist,
anti-inflammatory agent, an anti-IgE antibody (e.g., omalizumab)
and/or epinephrine. In some embodiments, the formulation is
administered with at least one membrane stabilizing agent (e.g.,
cromolyn). The membrane stabilizing agent acts to stabilize the
membranes of mast cells so that they cannot release molecules that
induce anaphylaxis.
VI. Examples
[0161] The present technology may be better understood by reference
to the following non-limiting examples. The following examples are
presented in order to more fully illustrate certain embodiments and
should in no way be construed, however, as limiting the broad scope
of the present technology. While certain embodiments of the present
technology have been shown and described herein, it will be obvious
that such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions may occur to those skilled
in the art without departing from the embodiments; it should be
understood that various alternatives to the embodiments described
herein may be employed in practicing the methods described
herein.
[0162] In the examples, egg white protein and formulations
comprising egg white protein are tested for stability under various
conditions. The total egg protein, ovomucoid protein, ovalbumin
protein, and/or lysozyme protein in the egg protein formulations
provided herein may be considered "stable" if its concentration is
.+-.10% the original concentration of such protein(s) in the egg
protein formulation immediately after manufacture.
Example 1
Evaluation of Egg White Powder
[0163] This example describes the physical characterization of the
Egg white protein. Egg white protein (Deb El Egg White Powder Lot
#PK049/2013) was used as the source of egg protein and was
physically characterized by visual inspection for appearance and
flow. Particle size studies were also performed using mesh filters.
The egg white protein was found to have the following properties
(see Table 4):
TABLE-US-00004 TABLE 4 Egg white powder properties: Appearance
(visual analysis) Color Off white Texture Fine powder Flowability
Cohesive Flow Studies Bulk Density 0.328 g/ml Tap Density 0.531
g/ml Carr Index 38.23 (>25 = poor flowability) Particle Size
Distribution 100% < 100 mesh (150 microns) ~82% < 200 mesh
(75 microns)
[0164] Dynamic vapor sorption analyses (DVA) were performed to
determine the conditions under which egg white protein moisture
content increases significantly (potentially decreasing flow and
protein stability), and to serve as a baseline for comparison with
the egg protein formulations provided herein. Deb El Egg White
Powder Lot #PK049/2013 was used and found to have the properties
listed in Table 5 below. Further, as shown in FIGS. 1-3, the rate
of change in the mass of the sample (rate of moisture uptake)
increased significantly at values above 50% relative humidity. As
shown in Table 12, at 75% relative humidity, the egg protein
formulations provided herein take up less moisture than egg white
protein alone.
TABLE-US-00005 TABLE 5 Deb El Egg White Powder properties Moisture
Testing Dynamic Vapor Sorption *<10% Change in Mass @ Relative
Humidity .ltoreq.40% LOD Results for Open Sample at Initial LOD
7.04% Percent Change Ambient Conditions for 4 days Final LOD 9.83%
in LOD = 39.63% Protein Integrity Assay Peak # Retention Time (min)
% Area Under the Curve 1 28.4 8.50 2 29.9 9.91 3 31.1 10.83 4 33.5
66.68 5 41.4 4.08 Total Protein Assay Anhydrous value 91.9% Use as
value 85.71%
Example 2
Forced Degradation of Egg white powder
[0165] This example describes tests to determine the stability of
egg white protein in the presence of acids, bases, peroxide, light
and heat. Acid, base, peroxide and heat were used to stress the egg
white protein solutions and the stability was evaluated. Evaluation
of the degradation of the samples was based on a chromatographic
assay of egg white protein in the stressed samples as compared to
unstressed samples.
[0166] The acid stressed sample was evaluated as follows: Egg white
protein was dissolved in H.sub.2O to form a 1.0 mg/mL solution and
exposed to 1.0 N HCl to create an acid stressed sample. The acid
stressed sample, an untreated sample containing only 1 mg/ml
untreated egg white protein solution, and an acid control
containing 1 N HCl were allowed to stand overnight at 4.degree. C.
At the end of the time period the test solution was neutralized
with 1.0 mL of 0.1 N NaOH and extracted for preparation of
chromatography.
[0167] The base stressed sample was prepared as follows: Egg white
protein was dissolved in H.sub.2O to form a 1.0 mg/mL solution and
exposed to 1.0 N NaOH to create a base stressed sample. The base
stressed sample, an untreated sample containing only 1 mg/ml
untreated egg white protein solution, and a base control containing
1 N NaOH were allowed to stand overnight at 4.degree. C. At the end
of the time period the test solution was neutralized with 1.0 mL of
0.1 N HCl and extracted.
[0168] The peroxide stressed sample was prepared as follows: Egg
white protein was dissolved in H.sub.2O to form a 1.0 mg/mL
solution and exposed to 3% hydrogen peroxide to create a peroxide
stressed sample. The peroxide stressed sample, an untreated sample
containing only 1 mg/ml untreated egg white protein solution, and a
peroxide control containing [3% hydrogen peroxide] were allowed to
stand overnight at 4.degree. C.
[0169] The heat stressed sample was prepared as follows: Egg white
protein was dissolved in H.sub.2O to form a 1.0 mg/mL solution and
exposed to 70.degree. C. overnight to create a heat stressed
sample. An unheated sample containing 1 mg/ml unheated egg white
protein solution, and a control were allowed to stand overnight at
4.degree. C.
[0170] The light exposed sample was prepared as follows: Egg white
protein was dissolved in H.sub.2O to form a 1.0 mg/mL solution and
exposed to light overnight to create a light exposed sample. A
sample containing 1 mg/ml egg white protein solution stored in the
dark, and a control were allowed to stand overnight at 4.degree.
C.
[0171] The samples from the degradation studies were analyzed using
an HPLC System (Size Exclusion Chromatography--SEC) under the
following conditions: Column (two in series): Phenomenex Yana SEC
2000, 3 .mu.m, 300.times.7.8 mm with the following reagents and
conditions: Mobile Phase: 0.05 M Na2HPO4, 0.05 M NaH2PO4, 0.15 M
NaCl, pH 6.8; Flow rate: 0.5 mL/min; Injection volume: 20 .mu.L;
Run time: approximately 60 minutes; Detector: UV 220 nm; Pump mode:
Isocratic; Column temperature: Ambient; and Needle wash: Water.
[0172] The results in FIGS. 4-8 showed no interfering peaks in the
control preparations. Peaks were evaluated qualitatively, and as
shown in FIGS. 4-8, significant degradation occurred with exposure
to acid, base, and heat, whereas the egg white protein solutions
were demonstrated to be less sensitive to peroxide and light.
Example 3
Identification and Quantification of Egg White Proteins
[0173] In this example, several assays were developed in order to
facilitate the quantification of the three major egg protein
allergens, Ovomucoid, Ovalbumin, and Lysozyme. The assays tested
were ELISA assays, size exclusion chromatography methods, and
immunoblot techniques.
[0174] The ELISA assay was performed as follows: Coating Antigen
was incubated overnight at 4.degree. C., then washed 3.times. with
PBS w/0.05% Tween 20. Then the antigen was blocked with 1% BSA in
PBS for 1 hour at 40.degree. C. and washed 3X with PBS w/0.05%
Tween 20. The primary antibody was added and incubated for 1.5
hours at 40.degree. C. and washed 3X with PBS w/0.05% Tween 20. The
Secondary Antibody (1:10000 for Ovomucoid and Ovalbumin, 1:5000 for
Lysozyme, no dilution) was added and incubated for 1.5 hours at
40.degree. C. The detection substrate was added and incubated until
there was a color change at room temperature, at which point the
stop solution was added. Absorbance was read at 450 nm using an
automated microtiter plate ELISA reader. An example of a microtiter
plate is shown below as Table 6:
TABLE-US-00006 TABLE 6 microtiter dilutions ##STR00001##
[0175] The ELISA assay was performed using various dilutions for
each protein (represented by different lines in FIGS. 9-15) at
varying primary antibody dilutions and was used to determine the
optimal conditions for each reagent, such that the amount of each
egg protein can be determined from the most sensitive (steepest
slope) portion of the titration curve. Primary antibody
concentration versus absorbance is shown in FIGS. 9-15 for each
dilution of each protein. The secondary antibody concentration was
1:5000 for all Lysozyme samples and 1:10000 for all Ovomucoid and
Ovalbumin samples, unless otherwise indicated.
[0176] A Size Exclusion Chromatography (SEC) assay using individual
egg proteins as well as blended powders was conducted under the SEC
conditions used in the forced degradation analysis of Example 2.
FIG. 16 shows the chromatogram overlays of Chicken Egg Albumin
(.about.0.4 mg/mL), Trypsin Inhibitor (.about.0.4 mg/mL), Lysozyme
(.about.0.4 mg/mL) and Egg White Protein (.about.1.0 mg/mL) using
two Phenomenex Yana 2000 SEC columns in series. FIG. 17 shows the
chromatogram overlays of Egg White Protein Placebo (Starch 1500),
Egg White Protein Standard (.about.0.02 mg/mL protein), Placebo
spiked with Egg White Protein (.about.0.02 mg/mL protein) and Egg
White Protein Standard prepared with Placebo supernatant.
[0177] A Size Exclusion Chromatography (SEC) assay using egg
protein in the different solvents shown in Table 7 was conducted
under the SEC conditions used in Example 2.
TABLE-US-00007 TABLE 7 1 mg/mL Egg Protein Peak Areas using Size
Exclusion Chromatography Diluent Unk 01 Unk 02 Unk 03 Total Area 50
mM NaPO4 ND 16868750 40301 16909051 50 mM NaPO4 w/0.1% SDS ND
14912834 80028 14992862 50 mM KPO4 ND 17161823 19266 17181089 50 mM
KPO4 w/0.1% ND 16311466 23669 16335135 Tween 20 200 mM NaPO4 ND
16873871 28161 16902032 200 mM NaPO4 w/0.1% SDS 43318 16537051 ND
16580369 200 mM KPO4 ND 17276749 206260 17483009 200 mM KPO4 w/0.1%
ND 17564491 144179 17708670 Tween 20 PBS 50121 18147730 57944
18255795 PBS w/0.1% SDS ND 12551258 129076 12680334 PBS w/0.1%
Tween 20 ND 16270054 181413 16451467 ND = Not Detected
[0178] FIG. 18 shows the chromatogram overlays of 1 mg/mL Egg White
Protein diluted with PBS solutions using size exclusion
chromatography. FIG. 19 shows the chromatogram overlays of 1 mg/mL
Egg White Protein diluted with 50 mM Phosphate Buffer solutions
using size exclusion chromatography. FIG. 20 shows the chromatogram
overlays of 1 mg/mL Egg White Protein diluted with 200 mM Phosphate
Buffer solutions using size exclusion chromatography. Nitrogen
Combustion Protein Quantification (Dumas Method Combustion) was
performed as follows: Dumatherm.RTM. Combustion assays permit
quantification of relatively small amounts of protein in a sample.
Such an assay was performed in which egg white protein samples were
combusted to determine the amount of N2 in a sample. The samples
were combusted using an O2 flow rate of 1.4 mL per mg of sample and
about 140 mg EDTA standard amount. The amount of N2 was used to
determine the total protein content in the sample (data in Table 8)
using the following calculation: Assay (%LC)=(%
Nitrogen-CF).times.TCFW/LC.times.5.46/100.times.100% Where: %
Nitrogen=% Nitrogen obtained in the Sample, CF=Nitrogen content
found in the Placebo, TCFW=Target Capsule Fill Weight, LC=Label
Claim of the Drug Product, 5.46=Conversion Factor fiom Nitrogen to
Protein Content. Linearity for blended egg protein protein was
shown in FIG. 21 between the sample weight (x axis) and nitrogen
peak area (y axis) as determined by the Dumatherm method. So as the
weight of each peak (protein) increases in molecular weight (later
elution time), the total nitrogen % area of each peak as plotted
increases in a proportional and linerar fashion (R.sup.2=0.97). The
results show the column method, as confirmed by the Dumatherm
method, can be used to both separate egg white proteins and to
determine total protein in a sample.
TABLE-US-00008 TABLE 8 Amount of N2 Sample % N2 Wt N.sub.2 Peak %
Corrected based on % Total (mg) area Nitrogen mg N Weight corrected
wt. Protein 20.57 1034 13.13 2.70 19.13 14.12 88.25 20.40 1027
13.16 2.68 18.97 14.15 88.41 40.48 5380 13.29 5.38 37.65 14.29
89.32 40.22 5336 13.27 5.34 37.40 14.27 89.17 80.32 37110 13.36
10.73 74.69 14.36 89.75 80.54 36940 13.25 10.67 74.90 14.25 89.04
100.21 46250 13.79 13.82 93.19 14.83 92.67 100.41 45950 13.66 13.71
93.38 14.69 91.79 120.47 53580 13.63 16.42 112.04 14.66 91.61
120.42 53410 13.59 16.36 111.99 14.61 91.30 200.93 83550 14.07
28.26 186.86 15.13 94.54 200.74 82740 13.91 27.92 186.69 14.95
93.47 280.45 105900 13.67 38.33 260.81 14.70 91.85 280.54 109100
14.22 39.89 260.90 15.29 95.56 Avg 91.19 RSD 2.53
[0179] The results show that any of the assays could be used to
identify and quantify the proteins in egg whites to provide
formulations having consistent levels of the egg proteins
ovalbumin, ovomucoid and lysozyme.
Example 4
Excipient Compatibility with Egg White Protein
[0180] The purpose of this compatibility study was to identify
excipients for a dry blend process which facilitate the goals of
processability on the encapsulation and sachet or pouch packaging
equipment, clean emptying of the contents from the capsule shells
or sachet packages, and chemical compatibility with the egg
proteins being dosed. As shown in Table 9, the excipients evaluated
in this study were grouped by functionality as filling agents,
diluents, glidants, colorants, capsule shell components, and
lubricants. Diluents and filling agents were evaluated at a ratio
of 1:1 versus the egg proteins (which constitute 91% of the egg
white protein by weight). Lubricants, glidants, colorants, and
capsule shell material were evaluated at a ratio of 10:1 (egg
protein versus the excipient or ingredient). Some excipients were
found to be incompatible with egg white protein in early
formulation development and were excluded. These are normal
accepted ranges/ratios based upon the functionality of each of the
class of excipients and or material tested.
[0181] Twelve excipients were evaluated including one filling
agent, three diluents, two glidants/anticaking agents, two
lubricants, and capsule shell material in four colors.
TABLE-US-00009 TABLE 9 Excipients Employed During Compatibility
Study Function- Manufacturer ality Excipient (Trade Name) Grade
Description Filling Mannitol Roquette NF Simple Organic Agent
(Pearlitol Diluent 300DC Diluent Partially Colorcon USP/NF Complex
Pregelatinized (Starch 1500) Organic Corn Starch Diluent Silicified
JRS Pharma USP Complex Microcrystalline PROSOLV Organic/ Cellulose
HD90) inorganic Co-processed Diluent Anhydrous Innophos USP
Inorganic Dicalcium (A-Tab) diluent Phosphate Glidant Colloidal
Silicon Cabot USP Glidant/ Dioxide (Cab-O-Sil Anticaking M5P) Agent
Talc Ultra USP Glidant/ Chemicals Anticaking (Ultra Talc Agent
4000) Lubricants Magnesium Mallinckrodt USP Lubricant Stearate
(vegetable source) Sodium Stearyl JRS Pharma USP Lubricant Fumarate
(Pruv) Capsule Clear Opaque Capsugel Vegetable Shell HPMC Capsule
(V-Caps) Source Shell Capsule Shell White Opaque Capsugel Vegetable
HPMC Capsule (V-Caps) Source Shell Capsule Shell Blue Opaque
Capsugel Vegetable HPMC Capsule (V-Caps) Source Shell Capsule
Shell
[0182] A. Excipients [0183] 1. Filling Agents
[0184] Filling Agents were evaluated. Mannitol was evaluated as a
simple organic filling agent as it has low hygroscopicity. [0185]
2. Diluents
[0186] Diluents representing three chemical categories were
evaluated: complex organic, inorganic, and combination co-processed
complex organic/inorganic (e.g., super-excipient). Microcrystalline
cellulose was evaluated as PROSOLV HD90 as the co-processed Prosolv
form can impart favorable processability even at low levels in the
formulation. Starch 1500 was evaluated as it performs the multiple
functions of imparting better flow and lubricity as well as being
particularly effective with moisture sensitive actives and low dose
geometric blending applications. A-Tab, an anhydrous inorganic
excipient, was evaluated for possibly providing protection against
moisture sorption. [0187] 3. Glidants
[0188] Cab-O-Sil (colloidal silicon dioxide) was included in the
formulation evaluation as a potential glidant/anticaking agent. Due
to its small particle size and large specific surface area,
colloidal silicon dioxide is known for flow enhancement
capabilities and moisture sequestering ability. Talc (USP) was
included in the formulation evaluation as an alternative to
Cab-O-Sil due to having similar glidant/anticaking properties to
colloidal silicon dioxide. [0189] 4. Lubricants
[0190] Magnesium stearate and sodium stearyl fumarate (e.g., PRUV)
were both evaluated as lubricants in the formulation evaluation.
Magnesium stearate is the most commonly used lubricant in the
pharmaceutical industry but, in some instances, can be susceptible
to non-compatibility with various drug molecules and, in some
instances, can be susceptible to "over-blending" which can affect
release rate. PRUV was also evaluated in this study. PRUV can
circumvent problems such as non-compatibility and over lubrication
which can increase the hydrophobicity of the blended material.
Thus, the bioavailability of certain actives may be improved by
using PRUV as the lubricant. [0191] 5. Capsule Shell
[0192] Hydroxypropyl Methyl Cellulose (HPMC) capsule shells were
selected for evaluation in this formulation study. HPMC capsule
shells are known to reduce the risk of protein/protein interactions
between gelatin capsule shells and the "active" egg proteins. The
compatibility study was performed with ground HPMC colored capsule
shells, using the natural, white and blue shells in the final
product. Sample matrices were prepared, representing the three
capsule shell colors, at a 10:1 (egg white powder: ground capsule
shell) ratio.
[0193] B. Formulations
[0194] Sixteen formulation matrices were evaluated (including a
control with egg white protein only) and as shown in Table 10. As
content uniformity can be a challenge in formulations that contain
extremely low levels of one or more ingredients, a geometric
blending technique was used to ensure the low level ingredient was
evenly distributed through the blends.
TABLE-US-00010 TABLE 10 Blend Formulations Excipient Name 1 2 3 4 5
6 7 8 9 10 11 12 13 14 Egg White 6.0 3.0 3.0 3.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 3.0 3.0 3.0 Powder.sup.1 Pearlitol 3.0 3.0 300DC
PROSOLV 3.0 3.0 HD90 Starch 1500 3.0 3.0 Cab-O-Sil 0.6 0.3 0.3 Talc
0.6 Magnesium 0.6 0.3 0.3 0.3 Stearate Pruv 0.6 Natural 0.6 0.3
capsule shells White capsule 0.6 0.3 shells Blue 0.6 0.3 capsule
shells Totals 6.0 6.0 6.0 6.0 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.9 6.9
6.6 .sup.1Formulation 1 contains only egg white protein as the
control in this study. Note: Formulations 2-8 contain egg white
protein at ratios of 1:1 or 10:1 to the excipients. Formulations
9-11 contain egg white protein at a ratio of 10:1 to the capsule
shell material. Formulations 12-13 contained blends of egg white
powder, excipient, Cab-O-Sil, magnesium stearate and capsule shell
material at a ratio of 10:10:1:1:1. Formulation 14 contained a
blend of egg white powder, excipient, magnesium stearate and
capsule shell material at a ratio of 10:10:1:1.
[0195] C. Process/Manufacturing
[0196] For each Egg white powder/Excipient ratio evaluated,
micro-blends of approximately 6 grams were prepared for evaluation
at controlled storage conditions. The sample matrices were used to
determine acceptable formulation excipients, which can then be
utilized in a common place manufacturing process. [0197] 1.
Procedure: Sample Preparation and Analysis
[0198] 1. Each of the formulation prototypes in Table 10 were
blended in a vial or in a mortar and pestle for approximately 10
minutes.
[0199] 2. Each of the micro-blends were then placed in clear,
borosilicate glass vials with Teflon lined screw-cap for
"packaging" the sample preparations.
[0200] 3. Samples were stored at 40.degree. C./75% Relative
Humidity storage conditions.
[0201] 4. Stability samples were tested chemically and for
appearance at time-zero, two-weeks, one-month, two-months and
three-months. Evaluation of appearance involved the color of the
powder, texture and determination of whether the material is
cohesive or free flowing.
[0202] 5. Biochemical analysis of the stability samples were
performed via HPLC analysis. Compatibility of the active material
with the individual excipients was established if no significant
changes in chromatography were seen between dry egg
protein/excipient blends and dry egg protein which was not exposed
to excipient. A sample of approximately 1.0 g was used for chemical
analysis.
[0203] 6. Loss on Drying was evaluated for the initial (T=O) sample
and at two months. A sample of approximately 2 g was used for LOD
testing. The sample was heated to below its melting point and the
moisture content (loss of water) measured.
[0204] D. Results Following 3-Month Excipient Compatibility
Study
[0205] Results of the 3 month excipient compatibility study is
presented herein. The study followed the parameters as set out
above and formulation samples were stored at 40.degree. C./75%
Relative Humidity.
[0206] As presented above in Table 10, blend formulations were
evaluated over a 3 month time period for a number of stability
characteristics. Protein integrity over time, as assessed by size
exclusion chromatography, is presented in Table 11 (% Area for each
protein peak) at 5 time points.
TABLE-US-00011 TABLE 11 Protein Integrity (reported data, excludes
placebo peaks) Assay Results (Reported as percent area by peak)
Sample Time Peak # # Point 1 Peak # 2 Peak # 3 Peak # 4 Peak # 5 1
T = 0 8.50 9.91 10.83 66.68 4.08 2 W 9.99 11.07 10.56 66.89 1.49 1
M 10.30 11.14 10.17 63.71 4.67 2 M 14.85 14.20 10.44 60.51 3 M
15.03 13.27 9.53 62.17 2 T = 0 9.30 9.92 10.71 66.14 3.93 2 W 10.28
10.93 10.58 66.79 1.41 1 M 11.25 11.15 10.17 62.71 4.73 2 M 12.70
12.88 9.53 64.88 3 M 14.74 13.16 9.33 62.77 3 T = 0 8.55 9.91 10.74
66.87 3.91 2 W 10.36 11.06 10.59 66.72 1.27 1 M 11.01 11.25 10.05
63.12 4.57 2 M 13.36 12.98 9.64 64.02 3 M 14.71 13.02 9.50 62.77 4
T = 0 8.58 9.89 10.70 66.89 3.95 2 W 10.30 11.15 10.53 66.87 1.14 1
M 10.06 10.98 10.00 64.22 4.75 2 M 13.31 12.91 9.70 64.09 3 M 13.40
12.83 9.62 64.15 5 T = 0 8.36 9.89 10.48 67.40 3.86 2 W 9.54 10.74
10.60 68.08 1.04 1 M 9.42 10.71 10.22 65.02 4.64 2 M 12.72 12.82
9.82 64.64 3 M 14.04 13.09 9.66 63.21 6 T = 0 8.66 9.86 10.79 66.76
3.93 2 W 9.39 10.67 10.78 68.15 1.00 1 M 9.25 10.70 10.29 65.12
4.63 2 M 13.06 12.75 9.87 64.32 3 M 14.02 13.00 9.65 63.33 7 T = 0
8.66 9.86 10.75 66.80 3.92 2 W 9.34 10.64 10.87 68.23 0.91 1 M 8.80
10.36 10.50 65.80 4.54 2 M 11.96 12.17 10.23 65.64 3 M 12.35 12.70
10.00 64.94 8 T = 0 8.34 9.81 10.77 67.09 3.99 2 W 8.95 10.50 10.83
68.82 0.90 1 M 8.94 10.30 10.53 65.72 4.52 2 M 11.57 12.25 10.20
65.98 3 M 12.23 12.57 9.96 65.24 9 T = 0 8.74 9.80 10.93 66.47 4.06
2 W 9.60 10.68 10.89 68.04 0.79 1 M 9.88 10.58 10.49 64.51 4.54 2 M
12.59 12.55 10.02 64.84 3 M 13.04 12.90 9.84 64.21 10 T = 0 8.94
9.88 10.78 66.43 3.97 2 W 9.51 10.77 10.92 68.09 0.72 1 M 8.77
10.35 10.29 65.80 4.79 2 M 12.78 12.71 10.02 64.50 3 M 13.56 12.89
9.84 63.70 11 T = 0 8.97 9.88 10.78 66.41 3.96 2 W 9.72 10.77 10.88
67.98 0.64 1 M 10.32 10.90 10.44 63.83 4.51 2 M 13.21 12.83 9.86
64.10 3 M 13.00 12.92 9.73 64.35 12 T = 0 9.32 10.02 10.41 66.41
3.84 2 W 8.34 10.80 10.33 70.05 0.47 1 M 6.71 10.34 9.74 68.35 4.87
2 M 13.61 13.03 9.32 64.04 3 M 13.09 12.97 9.00 64.94 13 T = 0 8.22
9.96 10.22 67.83 3.78 2 W 9.22 11.07 10.39 68.89 0.43 1 M 9.98
11.09 10.04 64.41 4.48 2 M 13.49 13.00 9.45 64.06 3 M 13.51 13.37
9.75 63.36 14 T = 0 7.99 9.73 10.80 67.56 3.91 2 W 9.31 10.98 10.72
68.57 0.40 1 M 9.23 10.68 10.25 65.33 4.51 2 M 13.15 12.84 9.81
64.20 3 M 14.16 13.01 9.51 63.32
[0207] At T=0, .SIGMA.4-6=98.07. Range of acceptability is +/-2% of
initial, giving range of 96.11-100.03. .SIGMA.4-6 for all samples
fall within this range.
[0208] Appearance observations include color, texture and
flowability. Loss on Drying was tested at T=0 and at the 2 month
time point. Appearance and Loss on Drying data are presented in
Table 12.
TABLE-US-00012 TABLE 12 Appearance and Loss on Drying Cohesive or
Sample # Time Color Texture Free-flowing % LOD .DELTA. LOD 1 T = 0
off white fine powder cohesive 9.9 1 M off white fine powder
cohesive 2 M off white fine powder cohesive 11.92 2.02 3 M off
white fine powder cohesive 2 T = 0 off white fine powder cohesive
4.3 1 M off white fine powder cohesive 2 M off white fine powder
cohesive 6.82 2.52 3 M off white fine powder cohesive 3 T = 0 off
white fine powder cohesive 6.2 1 M off white fine powder cohesive 2
M off white fine powder cohesive 9.00 2.80 3 M off white fine
powder cohesive 4 T = 0 off white fine powder cohesive 9.3 1 M off
white fine powder cohesive 2 M off white fine powder cohesive 11.47
2.17 3 M off white fine powder cohesive 5 T = 0 off white fine
powder cohesive 7.5 1 M off white fine powder cohesive 2 M off
white fine powder cohesive 10.95 3.45 3 M off white powder fine
powder with cohesive largewhiteclumps large white clumps 6 T = 0
off white fine powder free flowing 7.2 1 M off white fine powder
free flowing 2 M off white fine powder free flowing 11.00 3.80 3 M
off white fine powder cohesive 7 T = 0 off white fine powder
cohesive 7.6 1 M off white fine powder cohesive 2 M off white fine
powder cohesive 9.56 1.96 3 M off white fine powder cohesive 8 T =
0 off white fine powder cohesive 7.4 1 M off white fine powder
cohesive 2 M off white fine powder cohesive 8.98 1.58 3 M off white
fine powder cohesive 9 T = 0 off white fine powder cohesive 8.1
w/visible flecks 1 M off white fine powder cohesive w/visible
flecks 2 M off white fine powder cohesive 10.20 2.10 w/visible
flecks 3 M off white fine powder cohesive w/visible flecks 10 T = 0
off white fine powder cohesive 8.4 w/visible flecks 1 M off white
fine powder cohesive w/visible flecks 2 M off white fine powder
cohesive 10.85 2.45 w/visible flecks 3 M off white fine powder
cohesive w/visible flecks 11 T = 0 off white w/ fine powder
cohesive 9.0 blue specks w/visible flecks 1 M off white w/ fine
powder cohesive blue specks w/visible flecks 2 M off white w/ blue
fine powder cohesive 11.16 2.16 specks w/visible flecks 3 M off
white w/ blue fine powder cohesive specks w/visible flecks 12 T = 0
off white fine powder free flowing 5.8 1 M off white fine powder
free flowing 2 M off white fine powder free flowing 5.92 0.12 3 M
off white fine powder free flowing w/visible flecks 13 T = 0 off
white fine powder free flowing 8.6 1 M off white fine powder free
flowing 2 M off white fine powder free flowing 10.48 1.88 3 M off
white with fine powder small free flowing smallwhiteclumps white
clumps 14 T = 0 off white w/ fine powder free flowing 6.8 blue
specks w/visible flecks 1 M off white w/ blue fine powder free
flowing specks w/visible flecks 2 M off white w/ fine powder free
flowing 8.15 1.35 blue specks w/visible flecks 3 M off white w/
blue fine powder free flowing specks w/visible flecks
[0209] As shown in Table 12, three blends (12-14) were free-flowing
after 3 months. The results show that at 75% relative humidity, the
egg protein formulations (blends 12-14) take up less moisture than
egg white protein alone
Example 5
Egg Protein Formulations
[0210] Based on the compatibility study results in Example 4, the
formulations shown in Tables 13-16 were made and tested as
exemplary formulations. ProSolv SMCC 50 and ProSolv HD90 were
included as diluents, Mannitol was included as a filling agent,
Magnesium Stearate was included as a lubricant. However, other
diluents, filling agents and lubricants can be used with comparable
results. Example 6 provides the results of fill and recovery
testing for the exemplary formulations.
TABLE-US-00013 TABLE 13 Formulation for 0.2 mg capsules Item #
Ingredient % w/w mg/dose 1 EWP Pre-blend Lot 14004 (1.78% 7.15 11.3
protein) 2 ProSolv SMCC 50 36.58 57.8 3 ProSolv HD90 45.76 72.3 4
Mannitol 200SD 10.00 15.8 5 Magnesium Stearate 0.50 0.79 total 100
158
TABLE-US-00014 TABLE 14 Formulation for 1.0 mg capsules Item #
Ingredient % w/w mg/dose 1 EWP Preblend Lot 14008 (1.86% 35.85
53.77 Protein) 2 Prosolv SMCC 50 26.15 39.23 3 Prosolv HD90 27.51
41.26 4 Mannitol 200 SD 10.0 15.0 5 Magnesium Stearate 0.5 0.75
Total 100 150
TABLE-US-00015 TABLE 15 Formulation for 10.0 mg capsules Item #
Ingredient % w/w mg/dose 1 Egg White Powder 2.57 12.2 (10.0)
(81.85% protein) 2 ProSolv SMCC 50 38.46 182.7 3 ProSolv HD90 48.47
230.2 4 Mannitol 200SD 10.00 47.5 5 Magnesium Stearate 0.50 2.4
Total 100 475
TABLE-US-00016 TABLE 16 Formulation for 100.0 mg capsules Item #
Ingredient % w/w mg/dose 1 Egg White Powder 25.73 122.2 (100.0)
(81.85% protein) 2 ProSolv SMCC 50 28.21 134.0 3 ProSolv HD90 35.56
168.9 4 Mannitol 200SD 10.00 47.5 5 Magnesium Stearate 0.50 2.4
Total 100 475
Example 6
Fill and Recovery Analyses
[0211] The formulations in Tables 13-16 were tested for fill and
recovery when formulated in a capsule. The capsule fill and
recovery was evaluated for 4 dosage strengths of egg white protein:
0.2 mg, 1.0 mg, 10.0 mg and 100.0 mg. For each dosage strength, 100
capsules were made using the Pro-Fill.RTM. hand encapsulation unit.
The acceptance range for each set of capsules was .+-.3% of the
target fill weight from the formulation. All capsules used were
weight-checked to ensure they were within the acceptance range.
Recovery data is presented in Tables 17-20 for 0.2, 1.0, 10.0 and
100.0 mg capsules, respectively.
TABLE-US-00017 TABLE 17 Fill Recovery 0.2 mg Capsules Fill Mass
Total Weight Recovered Weight of % Recovery of Capsule per capsule
Empty per Capsule # (mg) (mg) Shell (mg) capsule 1 209 157 49 98.1
2 203 151 50 98.7 3 204 155 48 99.4 4 202 151 48 98.1 5 204 152 51
99.3 6 204 152 51 99.3 7 207 154 50 98.1 8 201 153 48 100.0 9 201
150 50 99.3 10 203 154 47 98.7 Avg 203.8 152.9 49.2 98.9 SD 2.5 2.1
1.4 0.7 RSD 1.2 1.4 2.8 0.7 Target (mg): 158 Average Fill Mass
Recovered 96.8 as % of Target
TABLE-US-00018 TABLE 18 Fill Recovery 1.0 mg Capsules Fill Mass
Recovered Weight of Total Weight of per capsule Empty % Recovery
Capsule # Capsule (mg) (mg) Shell (mg) per capsule 1 191 146 44
99.3 2 191 143 47 99.3 3 192 145 45 98.6 4 192 146 44 98.6 5 193
146 45 98.6 6 191 144 44 98.0 7 191 146 44 99.3 8 191 143 46 98.6 9
191 143 45 97.9 10 191 145 45 99.3 Avg 191.4 144.7 44.9 98.8 SD 0.7
1.3 1.0 0.5 RSD 0.4 0.9 2.2 0.5 Target (mg): 150 Average Fill Mass
Recovered as 96.5 % of Target
TABLE-US-00019 TABLE 19 Fill Recovery 10.0 mg Capsules Fill Mass
Total Weight Recovered Weight of of Capsule per capsule Empty %
Recovery Capsule # (mg) (mg) Shell (mg) per capsule 1 598 470 126
99.6 2 589 463 124 99.6 3 587 459 126 99.6 4 594 465 128 99.8 5 599
472 125 99.6 6 599 469 128 99.6 7 589 463 126 100.0 8 588 459 128
99.8 9 592 463 128 99.8 10 596 470 125 99.8 Avg 593.1 465.3 126.4
99.7 SD 4.7 4.7 1.5 0.1 RSD 0.8 1.0 1.2 0.1 Target (mg): 475
Average Fill Mass Recovered as 98.0 % of Target
TABLE-US-00020 TABLE 20 Fill Recovery 100.0 mg Capsules Fill Mass
Recovered per Weight of Total Weight of capsule Empty % Recovery
Capsule # Capsule (mg) (mg) Shell (mg) per capsule 1 592 463 123
98.7 2 599 469 128 99.6 3 591 465 122 99.1 4 588 463 123 99.6 5 601
474 123 99.2 6 597 472 121 99.2 7 586 462 121 99.4 8 598 474 122
99.6 9 587 453 132 99.6 10 584 460 121 99.4 Avg 592.3 465.5 123.6
99.3 SD 6.1 6.8 3.6 0.3 RSD 1.0 1.5 2.9 0.3 Target (mg): 475
Average Fill Mass Recovered as 98 % of Target
[0212] The recovery of the material from the capsules for the
formulations for the 0.2 mg, 1.0 mg, 10.0 mg, and 100.0 mg capsules
was between 98 and 100% for all capsules tested. This shows that
the formulations provided in Tables 13-16 can be used effectively
for the production of capsulized pharmaceuticals containing egg
white proteins.
Example 7
Blend and Content Uniformity Analyses
[0213] Blend and content uniformity analyses were conducted on
formulations based on those in Tables 13-16 as follows: The common
egg white protein powder (EWP) pre-blend shown in Table 21 was
created using Michael Foods EWP, lot 1248944. The common blend was
then diluted to give three dosage strengths (0.2 mg, 1.0 mg, and
10.0 mg). Geometric dilutions were used in the common blend as well
as the dilutions for the 0.2 mg and 1.0 mg dosage strengths.
Geometric dilution or geometric blending is a technique used in
mixing two ingredients of unequal quantities; one begins with the
smallest quantity and adds an equal quantity of the ingredient
having the larger amount. The process then continues until all of
the ingredients are used.
TABLE-US-00021 TABLE 21 Common Pre-blend Formulation Formulation
for Batch 054-MFG-14008 (Common Pre-blend) Item Concentration No.
Ingredient (% w/w) Amount/Batch (g) 1 Egg White Protein (Powder)
*1.78 (2.18) 32.12 (39.24) 2 Prosolv SMCC 50 43.04 774.76 3 Prosolv
HD90 54.78 986.00 Total 100 1800 *Based on egg white protein powder
containing 81.85% protein. (Blend was originally formulated based
on EWP containing 85.71% protein, which would have given the
pre-blend a protein concentration of 1.86%. The tables have been
adjusted to reflect the actual protein content.)
[0214] The "use as" protein content of Michael Foods EWP was found
to be 81.85%. Because this information was not available at the
time of blending, the original formulations were based on the
slightly higher protein content of the Deb El material, 85.71%. All
formulations were adjusted to reflect the Michael Food values. The
adjusted formulations for all batches are shown in Tables
22-24.
TABLE-US-00022 TABLE 22 Formulation for Batch 054-MFG-14009A
(0.2*/0.19 mg** Protein) Item Concentration Adjusted Amount/ No.
Ingredient (% w/w) mg/Dose mg/Dose) Batch (g) 1 EWP Pre-blend Lot
14008 (*1.86% 7.17 *10.75 **10.75 107.55 protein/**1.78% protein)
(0.2 mg) (0.19 mg) 2 ProSolv SMCC 50 36.60 54.90 54.90 549.00 3
ProSolv HD90 45.73 68.60 68.60 685.95 4 Mannitol 200SD 10.00 15.00
15.00 150.00 5 Magnesium Stearate 0.50 0.75 0.75 7.50 Total 100 150
150 1500 *Based on egg white powder containing 85.71% protein.
**Based on egg white powder containing 81.85% protein. (Blend was
originally formulated based on EWP containing 85.71% protein and
pre-blend concentration of 1.86% to give 0.2 mg protein per dose.
The adjusted column in the tables reflects the actual protein
content.)
TABLE-US-00023 TABLE 23 Formulation for Batch 054-MFG-14010B
(1.0*/0.96 mg** Protein) Item Ingredient Concentration mg/Dose
Adjusted Amount/ 1 EWP Pre-blend Lot 14008 (*1.86% 35.85 *53.77
**53.77 537.7 protein/**1.78% protein) (1.0 mg) (0.96 mg) 2 ProSolv
SMCC 50 26.15 39.23 39.23 392.2 3 ProSolv HD90 27.51 41.26 41.26
412.6 4 Mannitol 200SD 10.0 15.0 15.0 150.0 5 Magnesium Stearate
0.5 0.75 0.75 7.5 Total 100 150 150 1500 *Based on egg white powder
containing 85.71% protein **Based on egg white powder containing
81.85% protein. (Blend was originally formulated based on EWP
containing 85.71% protein and pre-blend concentration of 1.86% to
give 1.0 mg protein per dose. The adjusted column in the tables
reflects the actual protein content.)
TABLE-US-00024 TABLE 24 Formulation for Batch 054-MFG-14011C
(10.0*/9.55mg** Protein) Item Ingredient Concentration mg/Dose
Adjusted Amount/ 1 EWP Pre-blend Lot 14008 (*1.86% 89.6 *537.7
**537.7 474.9 protein/**1.78% protein) (10.0 mg) (9.55 mg) 2
Mannitol 200SD 10.0 60.0 60.0 53.0 3 Magnesium Stearate 0.4 2.3 2.3
2.1 Total 100 600 600 530 *Based on egg white powder containing
85.71% protein. **Based on egg white powder containing 81.85%
protein. (Blend was originally formulated based on EWP containing
85.71% protein and pre-blend concentration of 1.86% to give 10.0 mg
protein per dose. The adjusted column in the tables reflects the
actual protein content.) Blend was less dense than anticipated and
did not fill at target weight. Label claim was adjusted for this
batch. (Target fill weight was 600 mg. Actual fill weight was 478
mg. Adjusted label claim was 8 mg.)
[0215] All blends were thieved for blend uniformity testing. Blend
uniformity measures the uniformity of the blending method. It is
important that the active ingredient(s) are uniformly blended in
the formulation. A 1.0 cc sample thief was used to pull samples
from 10 unique regions of the blender. The thieved samples were
delivered directly into tared, labeled specimen cups. The weight of
the sample was determined as the difference between the empty and
filled cups. Analytical testing utilized the entire sample rather
than a portion of the thieved material. Capsule shells were
hand-filled using appropriately sized ProFill systems.
[0216] An analysis of egg protein content and blend uniformity was
conducted using size exclusion chromatography as provided in
Example 2. The calculations used to quantify the content
uniformity, the blend uniformity, and the total protein (assay for
egg white protein) from the chromatogram overlays are as
follows:
[0217] Content Uniformity (% Label Claim)
% Label Claim (%LC)=(RU/RS).times.CSTD.times.(VSPL/LC)'100
where: [0218] Ru=Area of Egg White Protein in the Sample [0219]
Rs=Average Egg White Protein Peak Area in all Standards [0220]
Cstd=Working Standard Concentration (mg/mL) [0221] Vspl=Volume of
Working Sample [0222] LC=Label claim [0223] 100=Conversion factor
to percentage
[0224] Blend Uniformity (% Label Claim)
% Label Claim
(%LC)=(R.sub.u/R.sub.s).times.C.sub.STD.times.(V.sub.SPL/Wt.sub.SPL).time-
s.(TCFW/LC).times.100
where: [0225] R.sub.u=Area of Egg White Protein in the Sample
[0226] R.sub.s=Average Egg White Protein Peak Area in all Standards
[0227] C.sub.std=Working Standard Concentration (mg/mL) [0228]
V.sub.spl=Volume of Working Sample [0229] Wt.sub.spl=Weight of
Working Sample [0230] TCFW=Target Capsule Fill Weight [0231]
LC=Label claim [0232] 100=Conversion factor to percentage
[0233] Assay for Egg White Protein
Assay=(R.sub.u/R.sub.s).times.C.sub.STD.times.(V.sub.sample/Wt.sub.sampl-
e).times.(ACFW/LC).times.100
where: [0234] R.sub.u=Area of Egg White Protein in the Sample
[0235] R.sub.s=Average Egg White Protein Peak Area in all Standards
[0236] C.sub.STD=Working Standard Concentration (mg/mL) [0237]
V.sub.Sample=Volume of the Working Sample (mL) [0238]
Wt.sub.Sample=Weight of Egg White Protein in the Sample (mg) [0239]
ACFW=Average Capsule Fill Weight [0240] LC=Label claim [0241]
100=Conversion factor to percentage
[0242] FIG. 22 shows the chromatogram overlay of egg white protein
standard (bottom) and 0.2 mg capsule Content Uniformity sample
(top). FIG. 23 shows the chromatogram overlay of egg white protein
standard (bottom) and 1.0 mg capsule Content Uniformity sample
(top). FIG. 24 shows the chromatogram overlay of egg white protein
standard (bottom) and 10.0 mg capsule content uniformity sample
(top). Blend analysis data for all batches is presented in Tables
25-28. Uniformity of content is a pharmaceutical analysis technique
for the quality control of capsules or tablets. Multiple capsules
or tablets are selected at random and a suitable analytical method
is applied to assay the individual content of the active ingredient
in each capsule or tablet. The preparation complies if not more
than one (all within limits) individual content is outside the
limits of 85 to 115% of the average content and none is outside the
limits of 75 to 125% of the average content. The results show that
from a uniformity perspective, all of the Blends were acceptable
with <.about.5% RSD.
[0243] Content uniformity and assay results for the filled capsules
are presented in Tables 29-31. Content Uniformity was acceptable
for the 0.2 mg and 1.0 mg capsules with RSD <.about.4%. However,
content uniformity for the 10.0 mg capsules had an RSD of 8%. This
is significantly more variability than the blend alone.
TABLE-US-00025 TABLE 25 Blend Analysis, Batch 054-MFG-14008 (Common
Pre-blend) % % Sample Recovery Sample Recovery 1 94.84 6 97.71 Mean
96.37 2 103.72 7 96.07 Std Dev 3.15 3 96.56 8 96.29 % RSD 3.26 4
97.58 9 95.70 5 92.15 10 93.05
TABLE-US-00026 TABLE 26 Blend Analysis, Batch 054-MFG-14009A (0.2
mg) Sample % Recovery Sample % Recovery 1 81.91 6 80.53 Mean 80.91
2 80.59 7 82.65 Std Dev 1.69 3 82.08 8 78.80 % RSD 2.08 4 77.81 9
81.76 5 80.01 10 83.00
TABLE-US-00027 TABLE 27 Blend Analysis, Batch 054-MFG-14010B (1.0
mg) Sample % Recovery Sample % Recovery 1 95.07 6 86.48 Mean 89.65
2 93.32 7 89.49 Std Dev 3.68 3 88.75 8 91.61 % RSD 4.10 4 93.04 9
84.07 5 89.65 10 84.98
TABLE-US-00028 TABLE 28 Blend Analysis, Batch 054-MFG-14011C (10.0
mg) Sample % Recovery Sample % Recovery 1 90.11 6 83.94 Mean 88.63
2 89.83 7 89.03 Std Dev 4.58 3 88.62 8 94.03 % RSD 5.17 4 91.09 9
90.62 5 77.80 10 91.24
TABLE-US-00029 TABLE 29 Content Uniformity, Batch 054-MFG-14009A
(0.2 mg) Sample % Recovery Sample % Recovery 1 99.53 6 90.78 Mean
93.80 2 90.28 7 91.55 Std Dev 3.99 3 92.84 8 88.07 % RSD 4.25 4
93.34 9 94.39 5 99.82 10 97.43 Assay: 80.11%
TABLE-US-00030 TABLE 30 Content Uniformity, Batch 054-MFG-14010B
(1.0 mg) Sample % Recovery Sample % Recovery 1 92.72 6 91.98 Mean
92.81 2 90.80 7 95.52 Std Dev 3.04 3 88.37 8 90.50 % RSD 3.28 4
96.02 9 89.78 5 97.55 10 94.89 Assay: 99.69%
TABLE-US-00031 TABLE 31 Content Uniformity, Batch 054-MFG-14011C
(10.0 mg) Sample % Recovery Sample % Recovery 1 99.40 6 78.63 Mean
98.66 2 105.59 7 103.35 Std Dev 7.83 3 102.57 8 96.45 % RSD 7.93 4
103.37 9 100.09 5 94.37 10 102.81 Assay: 103.46%
[0244] Content uniformity testing was conducted on newly filled
capsules of the blend made in Batch 054-002-14009A. The new
capsules are identified as Batch L0136-42.
[0245] The original blend in Batch 054-002-14009A was formulated
based on the protein content in Deb El Egg White Powder. The
results of the protein content of Michael Foods Egg White Protein
required the label claim to be adjusted down to 0.19 mg for this
batch. The formulation for the original Batch 054-002-14009 A is
shown in Table 32.
TABLE-US-00032 TABLE 32 Formulation for Batch 054-002-14009A
(0.2*/019 mg** Protein) Item Ingredient Concentration mg/Dose
Adjusted Amount/ 1 EWP 7.17 *10.75 **10.75 107.55 Pre-blend (0.2
mg) (0.19 mg) Lot 14008 (*1.86% protein/ **1.78% protein) 2 ProSolv
36.60 54.90 54.90 549.00 SMCC 50 3 ProSolv 45.73 68.60 68.60 685.95
HD90 4 Mannitol 10.00 15.00 15.00 150.00 200SD 5 Magnesium 0.50
0.75 0.75 7.50 Stearate Total 100 150 150 1500 *Based on egg white
powder containing 85.71% protein. **Based on egg white powder
containing 81.85% protein. (Blend was originally formulated based
on EWP containing 85.71% protein and pre-blend concentration of
1.86% to give 0.2 mg protein per dose. The adjusted column in the
tables reflects the actual protein content.)
[0246] The blend from the original batch was encapsulated at a
higher fill weight to give a true 0.2 mg dosage form. These
capsules are identified as Batch L0136-42. The adjusted formulation
is shown in Table 33. The content uniformity results are shown in
Tables 34-37.
TABLE-US-00033 TABLE 33 Formulation for Batch L0136-42 (0.2 mg
Protein) Ingredient Concentration mg/Dose Adjusted 1 EWP Pre-blend
Lot 14008 7.17 *10.75 11.32 (*1.86% protein/**1.78% (0.19 mg) (0.2
mg) protein) 2 ProSolv SMCC 50 36.60 54.90 57.81 3 ProSolv HD90
45.73 68.60 72.24 4 Mannitol 200SD 10.00 15.00 15.80 5 Magnesium
Stearate 0.50 0.75 0.79 Total 100 150 158 *Based on egg white
powder containing 85.71% protein. **Based on egg white powder
containing 81.85% protein.
TABLE-US-00034 TABLE 34 Content Uniformity, Batch 054-MFG-14009A
(0.19 mg) % % Sample Recovery Sample Recovery 1 99.53 6 90.78 Mean
93.80 2 90.28 7 91.55 Std Dev 3.99 3 92.84 8 88.07 % RSD 4.25 4
93.34 9 94.39 5 99.82 10 97.43 Assay: 80.11%
TABLE-US-00035 TABLE 35 Content Uniformity, Batch L0136-42 (0.2 mg)
(n = 10) % % Sample Recovery Sample Recovery 1 91.07 6 89.79 Mean
94.91 2 109.14 7 96.82 Std Dev 6.72 3 94.28 8 83.84 % RSD 7.08 4
98.05 9 91.35 5 98.09 10 96.71 Assay: 87.1%
TABLE-US-00036 TABLE 36 Content Uniformity, Batch L0136-42 (0.2
mg), (Repeated @ n = 20) % % Sample Recovery Sample Recovery 1
90.26 11 91.55 Mean 92.92 2 88.41 12 94.01 Std Dev 4.29 3 99.15 13
88.03 % RSD 4.62 4 99.31 14 93.01 5 89.05 15 93.47 6 88.40 16 94.26
7 103.92 17 91.22 8 92.65 18 95.06 9 89.86 19 95.06 10 94.57 20
87.12 Assay: 79.8%
TABLE-US-00037 TABLE 37 Content Uniformity, Batch L0136-42 (n = 30)
Mean 93.58 Std Dev 5.20 % RSD 5.55
[0247] Blend and content uniformity testing were conducted on
Batches 054-002-14012C and 054-002-14013D by blending the
formulations for 10-30 minutes in a blender and thieving a sample
at each time points. The tests were conducted on batches having 10
mg and 100 mg dosage strengths, respectively. Both batches
contained Michael Foods egg white protein powder from lot 4043W-3.
The 10 mg batch was processed as a geometric dilution while the 100
mg batch was processed as a direct blend. Formulations for the
batches are given in Tables 38 and 39. Blend and content uniformity
data for the 10 mg batch are presented in Tables 40 and 41. Blend
and content uniformity data for the 100 mg batch are presented in
Tables 42 and 43.
TABLE-US-00038 TABLE 38 Formulation for Batch 054-MFG-14012C Item #
Ingredient % w/w mg/dose g/batch 1 Egg White Powder (81.85% 2.57
12.2 (10.0) 38.55 protein) 2 ProSolv SMCC 50 38.46 182.7 576.90 3
ProSolv HD90 48.47 230.2 727.05 4 Mannitol 200SD 10.00 47.5 150.00
5 Magnesium Stearate 0.50 2.4 7.50 Total 100 475 1500 *Based on egg
white powder containing 81.85% protein.
TABLE-US-00039 TABLE 39 Formulation for Batch 054-MFG-14013D Item #
Ingredient % w/w mg/dose g/batch 1 Egg White Powder (81.85% 25.73
122.2 (100.0) 385.95 protein) 2 ProSolv SMCC 50 28.21 134.0 423.15
3 ProSolv HD90 35.56 168.9 533.40 4 Mannitol 200SD 10.00 47.5
150.00 5 Magnesium Stearate 0.50 2.4 7.50 Total 100 475 1500 *Based
on egg white powder containing 81.85% protein.
TABLE-US-00040 TABLE 40 Blend Analysis, Batch 054-MFG-14012C (10.0
mg) Sample % Recovery Sample % Recovery 1 89.87 6 98.96 Mean 97.58
2 86.97 7 94.69 Std Dev 7.38 3 94.33 8 94.76 % RSD 7.56 4 97.77 9
101.17 5 112.61 10 104.68
TABLE-US-00041 TABLE 41 Content Uniformity, Batch 054-MFG-14012C
Sample % Recovery Sample % Recovery 1 96.66 6 103.00 Mean 103.06 2
101.41 7 108.40 Std Dev 4.13 3 107.36 8 100.01 % RSD 4.01 4 108.23
9 98.24 5 104.89 10 102.43 Assay: 107.8%
TABLE-US-00042 TABLE 42 Blend Analysis, Batch 054-MFG-14013D (100.0
mg) Sample % Recovery Sample % Recovery 1 108.25 6 98.62 Mean
103.26 2 98.08 7 100.79 Std Dev 6.06 3 118.44 8 103.84 % RSD 5.87 4
100.06 9 102.38 5 100.86 10 101.27
TABLE-US-00043 TABLE 43 Content Uniformity, Batch 054-MFG-14013D
Sample % Recovery Sample % Recovery 1 100.22 6 101.47 Mean 101.21 2
102.87 7 98.33 Std Dev 1.51 3 100.43 8 101.69 % RSD 1.50 4 103.37 9
101.24 5 99.98 10 102.45 Assay: 101.6
[0248] The process study was conducted to identify how blended the
formulation was at different times of blending. The study used a
1.0 mg dosage strength as follows: Five sets of blend samples were
thieved: 1. Following geometric dilution of egg white powder with
SMCC50; 2. At 10 minutes of mix time with all diluents; 3. At 20
minutes of mix time with all diluents; 4. At 30 minutes of mix time
with all diluents and lubricant, with thieved sample size of
1-3.times. unit dose; 5. At 30 minutes of mix time with all
diluents and lubricant, with thieved sample size of 5-10.times.
unit dose. The results of the study showed that the formulations
were blended by 10 minutes time.
[0249] A swab recovery study was also conducted to look at
efficiency of removal from the blender. Two samples were collected,
one from each leg of the v-shell blender. The formulation of Batch
054-d02 14014B is shown in Table 44. Blend sampling recovery
results are presented in Tables 45-49. The swab recovery results
are presented in Table 50 and show that the recovery for this
formulation was excellent. The measurement of protein recovered was
a measurement of the total egg white protein. The results show that
the blended formulation was recovered very efficiently from the
blender.
TABLE-US-00044 TABLE 44 Formulation for 1 mg dose Item Ingredient
Concentration mg/Dose Amount/Batch 1 Egg White 0.80 1.20 (1.0) 12.0
Protein (EWP) 2 Prosolv SMCC 50 41.60 62.4 624.0 3 Prosolv HD90
47.10 70.65 706.5 4 Mannitol 200 SD 10.0 15.0 150.0 5 Magnesium 0.5
0.75 7.5 Stearate Total 100 150 1500 *Formula based on egg white
powder containing 83.3% protein.
TABLE-US-00045 TABLE 45 Blend Sampling, Step 8: Dilution with
SMCCSO Sample % Recovery 1 96.89 2 90.46 3 66.70 4 91.77 5 140.54 6
92.43 Ave 96.47 Std Dev 24.10 % RSD .24.98
TABLE-US-00046 TABLE 46 Blend Sampling, Step 10: At 10 Minutes Mix
Time Sample % Recovery 1 91.76 2 92.09 3 92.86 4 90.52 5 90.28 6
89.20 7 90.88 8 93.65 9 91.73 10 91.40 Ave 91.44 Std Dev. 1.29 %
RSD 1.41
TABLE-US-00047 TABLE 47 Blend Sampling, Step 12: At 20 Minutes Mix
Time Sample % Recovery 1 93.17 2 87.15 3 91.38 4 88.17 5 95.89 6
90.92 7 93.78 8 89.33 9 87.61 10 88.41 Ave 90.58 Std Dev 2.95 % RSD
3.26
TABLE-US-00048 TABLE 48 Blend Sampling, Step 15: At 30 Minutes Mix
Time, with Sample Size of 1-3X Unit Dose Sample % Recovery 1 84.35
2 83.56 3 83.40 4 82.93 5 83.31 6 87.34 7 103.47 8 82.34 9 84.87 10
81.90 Ave 85.75 Std Dev. 6.41 % RSD 7.48
TABLE-US-00049 TABLE 49 Blend Sampling, Step 15: At 30 Minutes Mix
Time, with Sample Size of 5-10X Unit Dose Sample % Recovery 1 87.17
2 87.22 3 87.57 4 87.74 5 87.84 6 87.89 7 87.93 8 88.56 9 88.09 10
86.53 Ave 87.65 Std Dev. 0.56 % RSD 0.64
TABLE-US-00050 TABLE 50 Swab Recovery Swab #1: Left Front Swab #1:
Right Back 0.069 mg protein recovered 0.084 mg protein recovered
Average protein recovered = 0.0765 mg Protein contained in blend =
9,996 mg Protein "lost" in blend process = 0.000765%
Example 8
High Dose Flow Recovery Analyses
[0250] High dose pouch (sachet) blends are typically used for doses
of more than 100 mg because they no longer fit within a capsule
(e.g., 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800
mg, 900 mg and 1000 mg). High does pouch blends are often put in a
pouch or sugar packet. The high dose pouch blends were analyzed for
flowability and fill recovery. The flowability and recovery are
studies to make sure that the capsule can be reproducibly filled by
automated encapsulation machines, and to optimize the efficiency by
which the capsule is easily emptied of powder by the subject.
Initial formulations contained only egg white powder and talc. The
flowability of these blends was very poor. The next set of
prototypes contained egg white powder, PROSOLV.RTM.HD90, mannitol
and magnesium stearate. The flowability of these blends was also
very poor. Mannitol was removed from the formulation for the third
set of prototypes. In the third set, two concentrations of talc
were investigated to determine if improved flow could be obtained
at levels low enough to avoid a negative impact on mouth feel. The
flowability of these blends was markedly better than the earlier
prototypes with a flow character of "passable".
[0251] The formulations for all prototypes are presented in Tables
51-53. Flow data for the prototypes are presented in Table 54. For
comparison, Table 55 shows the Scale of Flowability. Recovery data
are presented in Table 56.
TABLE-US-00051 TABLE 51 Formulations for Prototypes, Set A #
Ingredients % w/w g/batch 1 Egg powder 97.5 34.12 Talc 2.5 0.88
Total 100 35 2 Egg powder 95.0 33.25 Talc 5.0 1.75 Total 100 35 3
Egg powder 92.5 32.37 Talc 7.5 2.63 Total 100 35 4 Egg powder 90.0
31.5 Talc 10.0 3.5 Total 100 35
TABLE-US-00052 TABLE 52 Formulations for Prototypes, Set B #
Ingredients % w/w g/batch 5 Egg powder 58.34 87.51 PROSOLV .RTM.
36.16 54.24 Mannitol 5.0 7.5 Magnesium Stearate 0.5 0.75 Total 100
150 6 Egg powder 58.34 87.51 PROSOLV .RTM. 31.16 46.74 Mannitol
10.0 15.0 Magnesium Stearate 0.5 0.75 Total 100 150 7 Egg powder
58.34 87.51 PROSOLV .RTM. 26.16 39.24 Mannitol 15.0 22.5 Magnesium
Stearate 0.5 0.75 Total 100 150 8 Egg powder 58.34 87.51 PROSOLV
.RTM. 21.16 31.74 Mannitol 20.0 30.0 Magnesium Stearate 0.5 0.75
Total 100 150
TABLE-US-00053 TABLE 53 Formulations for Prototypes, Set C #
Ingredients % w/w g/batch 9 Egg powder 50.0 75.0 PROSOLV .RTM. 48.5
72.75 Talc 1.0 1.5 Magnesium Stearate 0.5 0.75 Total 100 150 10 Egg
powder 50.0 75.0 PROSOLV .RTM. 47.0 70.5 Talc 2.5 3.75 Magnesium
Stearate 0.5 0.75 Total 100 150
TABLE-US-00054 TABLE 54 Flowability, Recovery & Observations
Sample Carr's Hausner Prototype Set Number Index Ratio A 1 40.3
1.67 2 36.7 1.58 3 35.4 1.55 4 35.7 1.56 B 5 38.0 1.61 6 38.9 1.64
7 41.0 1.70 8 39.0 1.64 C 9 21.0 1.27 10 22.1 1.28
TABLE-US-00055 TABLE 55 Scale of Flowability Carr's Index (%) Flow
Character Hausner Ratio .ltoreq.10 Excellent 1.00-1.11 11-15 Good
1.12-1.18 16-20 Fair 1.19-1.25 21-25 Passable 1.26-1.34 26-31 Poor
1.35-1.45 32-37 Very poor 1.46-1.59 >38 Very, very poor
>1.60
TABLE-US-00056 TABLE 56 Pouch Recovery Set A Set B Set C Prototype
Recovery Prototype Recovery Prototype Recovery 1 94.8% 5 98.3% 9
97.5% 2 87.9% 6 97.9% 10 97.8% 3 93.7% 7 97.8% 4 95.4% 8 98.1%
[0252] As a result of the studies, a talc formulation was
determined to be more useful for flowability for high dose blends.
In one embodiment, a 1% talc formulation is used in the high dose
pouches. Approximately 1.2 g fill weight is used for the 500 mg
dosage strength and 2.4 g for the 1000 mg. The exact weights are
determined based on the protein content of the egg white powder
used in each batch.
Example 9
Six Month Stability Study
[0253] The stability of egg white protein capsules was analyzed for
6 months for 0.2 mg, 1.0 mg, 10 mg, and 100 mg capsules. The
exemplary formulations in Tables 57-60 were used to identify
general stability of the egg white protein formulations described
herein. All blends for this study were processed as GMP
demonstration batches using Michael Foods egg white powder (lot
4043W-3). The capsules used for this study were produced in the
following batches: D14089, 0.2 mg egg white protein capsules;
D14084, 1.0 mg egg white protein capsules; D14072, 10.0 mg egg
white protein capsules; and D14070, 100.0 mg egg white protein
capsules.
[0254] The stability protocol was as follows: Samples were stored
at 25.degree. C./60% RH and 30.degree. C./65% RH. The capsules were
packaged and stored in HDPE bottles with desiccant and CRC heat
sealed closures. The count per bottle was 10 for 0.2 mg in a 30 cc
bottle, 200 for 1.0 mg in a 150 cc bottle, 200 for 10.0 mg in a 500
cc bottle, and 200 for 10 mg in a 500 cc bottle. The number of
capsules tested for each test was as follows: Appearance and
Olfactory: n=10, LOD: 2g (14 capsules for 0.2, 1.0 mg/5 capsules
for 10, 100 mg); Potency of Ovomucoid (ELISA): n=10; Assay (protein
content by HPLC): n=10, Deliverable Mass: n=10, Content Uniformity:
n=20 (performed only at T=0), and Micro: 15g (100 of 0.2 and 1.0/32
of 10.0 and 100.0). The samples were tested for T=0 data when the
batches were produced. The samples were pulled from chambers at 1
M, 2 M, 3 M, and 6 M (and 11 M for Example 10). Each sample was
tested for appearance (shell and contents), Olfactory, LOD, potency
of ovomucoid, assay and deliverable mass. Content uniformity
testing was performed at T=0 and microbial limits testing was
performed at T=0 and T=12 M (see examples 1-8 for each test).
[0255] Formulations for the batches are given in Tables 57 through
60. Appearance and Odor are summarized in Tables 61 through 64.
Loss on Drying results are presented in Table 65. Potency of
Ovomucoid (ELISA) is presented in Table 66. Assay results are
presented in Table 67. Content Uniformity results are presented in
Table 68. Blend Uniformity results are presented in Table 69.
Deliverable Mass data are presented in Table 70.
TABLE-US-00057 TABLE 57 Formulation for EOIT; Batch D14089 (0.2 mg)
Item # Ingredient % w/w mg/dose g/batch 1 Egg White Powder 0.173
(0.160) 0.26 (0.24) 8.70 2 ProSolv SMCC 50 42.13 63.2 2106.5 3
ProSolv HD90 47.20 70.8 2360.0 4 Mannitol 200SD 10.00 15.0 500.00 5
Magnesium 0.50 0.75 25.0 Stearate Subtotal 100 150 5000 White
Opaque VCaps Plus, 3 47 Total 197 *Based on egg white powder
containing 81.85% protein. Includes a 5% overage of egg white
protein.
TABLE-US-00058 TABLE 58 Formulation for EOIT; Batch D14084 (1.0 mg)
Item # Ingredient % w/w mg/dose g/batch 1 Egg White Powder 0.84
(0.69) 1.26 (1.03) 42.0 2 ProSolv SMCC 50 41.56 62.34 2078.0 3
ProSolv HD90 47.10 70.65 2355.0 4 Mannitol 200SD 10.00 15.0 500.0 5
Magnesium Stearate 0.50 0.75 25.0 Subtotal 100 150 5000 Natural
Transparent VCaps Plus, 3 47 Total 197 *Based on egg white powder
containing 81.85% protein. Includes a 3% overage of egg white
protein.
TABLE-US-00059 TABLE 59 Formulation for EOIT; Batch D14072 (10.0
mg) Item # Ingredient % w/w mg/dose g/batch 1 Egg White Powder 2.57
12.2 (10.0) 128.5 (105.2) 2 ProSolv SMCC 50 38.46 182.7 1923 3
ProSolv HD90 48.47 230.2 2423.5 4 Mannitol 200SD 10.00 47.5 500.00
5 Magnesium Stearate 0.50 2.4 25.0 Subtotal 100 475 5000 Blue
Opaque VCaps Plus, 00 120 Total 595 *Based on egg white powder
containing 81.85% protein.
TABLE-US-00060 TABLE 60 Formulation for EOIT; Batch D14070 (100.0)
Item # Ingredient % w/w mg/dose g/batch 1 Egg White Powder 25.73
122.2 (100.0) 1286.5 (1000.0) 2 ProSolv SMCC 50 28.21 134.0 1410.5
3 ProSolv HD90 35.56 168.9 1778.0 4 Mannitol 200SD 10.00 47.5 500.0
5 Magnesium Stearate 0.50 2.4 25.0 Subtotal 100 475 5000 6 Sw
Orange VCaps Plus 120 Shell, 00 Total 595 *Based on egg white
powder containing 81.85% protein.
TABLE-US-00061 TABLE 61 Physical Characteristics of Batch 14089
(0.2 mg) Time point Appearance Odor T = 0 white capsule containing
white, free-flowing no distinct odor fine powder with few clumps T
= 1 M white capsule containing white, free-flowing no distinct odor
fine powder with few clumps T = 2 M white capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 3 M
white capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 4.5 M white capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 6 M
white capsule containing white, free-flowing no distinct odor fine
powder with few clumps
TABLE-US-00062 TABLE 62 Physical Characteristics of Batch 14084
(1.0 mg) Time point Appearance Odor T = 0 clear capsule containing
white, free-flowing no distinct odor fine powder with few clumps T
= 1 M clear capsule containing white, free-flowing no distinct odor
fine powder with few clumps T = 2 M clear capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 3 M
clear capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 4.5 M clear capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 6 M
clear capsule containing white, free-flowing no distinct odor fine
powder with few clumps
TABLE-US-00063 TABLE 63 Physical Characteristics of Batch 14072
(10.0 mg) Time point Appearance Odor T = 0 dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
1 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor T = 2 M dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
3 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor T = 4.5 M dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
6 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor
TABLE-US-00064 TABLE 64 Physical Characteristics of Batch 14070
(100.0 mg) Time point Appearance Odor T = 0 Burnt orange capsule
with gray bar on cap, black faint bar on body; off-white,
free-flowing fine powder acetic with few clumps acid odor T = 1 M
Burnt orange capsule with gray bar on cap, black faint bar on body;
off-white, free-flowing fine powder acetic with few clumps acid
odor T = 2 M Burnt orange capsule with gray bar on cap, black faint
bar on body; off-white, free-flowing fine powder acetic with few
clumps acid odor T = 3 M Burnt orange capsule with gray bar on cap,
black faint bar on body; off-white, free-flowing fine powder acetic
with few clumps acid odor T = 4.5 M Burnt orange capsule with gray
bar on cap, black faint bar on body; off-white, free-flowing fine
powder acetic with few clumps acid odor T = 6 M Burnt orange
capsule with gray bar on cap, black faint bar on body; off-white,
free-flowing fine powder acetic with few clumps acid odor
TABLE-US-00065 TABLE 65 Loss on Drying Batch Batch Batch Batch
D14089 D14084 D14072 D14070 (0.2 mg) (1 mg) (10 mg) (100 mg) Time T
= 0, 2.7% T = 0, 3.9% T = 0, 4.4% T = 0, 5.2% Condition 25/60 30/65
25/60 30/65 25/60 30/65 25/60 30/65 T = 1 M 3.7% 3.5% 5.1% 4.2%
5.3% 4.7% 5.8% 5.4% T = 2 M 3.5% 3.1% 4.2% 4.3% 4.6% 4.5% 5.5% 5.1%
T = 3 M 3.1% 3.3% 4.3% 4.3% 4.5% 4.5% 5.5% 5.2% T = 4.5 M 3.1% 3.5%
4.2% 4.4% 4.5% 4.5% 5.3% 5.3% T = 6 M 4.7% 4.1% 5.1% 4.9% 5.0% 4.7%
5.8% 5.6%
TABLE-US-00066 TABLE 66 Potency of Ovomucoid (ELISA) Batch Batch
Batch Batch D14089 D14084 D14072 D14070 Time (0.2 mg) (1 mg) (10
mg) (100 mg) Condition 25/60 30/65 25/60 30/65 25/60 30/65 25/60
30/65 T = 0 Data not available at T = 0; Method Development was
still in progress. T = 1 M 91.1 85.4 89.3 73.8 96.2 107.2 89.9
115.2 T = 2 M 41.3 42.2 49.9 51.5 81.8 96.3 100.1 106.1 T = 3 M
65.0 69.2 67.3 93.9 71.8 91.2 94.5 85.4 T = 4.5 M 69.3 76.6 88.9
81.3 90.2 94.8 90.3 96.8 T = 6 M 99* 83* 90** 93** 86** 84** 91**
73** 75** 63** *With place spiked standard **With regular
standard
TABLE-US-00067 TABLE 67 Assay (HPLC) Batch Batch Batch Batch D14089
D14084 D14072 D14070 Time (0.2 mg) (1 mg) (10 mg) (100 mg)
Condition 25/60 30/65 25/60 30/65 25/60 30/65 25/60 30/65 T = 0
95.5 100.0 102.4 110.0 T = 1 M 113.5 113.8 97.5 96.6 97.4 96.3
106.3 107.2 T = 2 M 87.9 85.9 96.6 94.9 97.9 96.2 104.3 101.8 T = 3
M 86.0 79.9 96.7 92.5 100.5 98.0 102.8 105.0 T = 4.5 M 83.5 78.8
88.3 89.3 93.3 89.3 97.5 99.6 T = 6 M 87.7 80.1 91.1 85.8 94.9 89.8
102.7 100.8
TABLE-US-00068 TABLE 68 Content Uniformity (Run only at T = 0)
Batch Batch Batch Batch D14089 D14084 D14072 D14070 (0.2 mg) (1 mg)
(10 mg) (100 mg) Range 88-102 86-112 99-106 104-118 Ave 93.39
101.74 102.25 110.01 % RSD 5.07 6.73 1.91 3.70
TABLE-US-00069 TABLE 69 Blend Uniformity (Run only at T = 0) Batch
Batch Batch Batch D14089 D14084 D14072 D14070 (0.2 mg) (1 mg) (10
mg) (100 mg) Range 76-84 94-100 98-105 105-117 Ave 79.21 94.64
101.39 108.55 % RSD 3.68 2.29 2.17 3.31
TABLE-US-00070 TABLE 70 Deliverable Mass (Report as Percent) Batch
Batch Batch Batch D14089 D14084 D14072 D14070 Time (0.2 mg) (1 mg)
(10 mg) (100 mg) Conditio 25/60 30/65 25/60 30/65 25/60 30/65 25/60
30/65 T = 0 Ave 100.5 100.2 100.1 100.1 T = 0 % RSD 0.4 0.3 0.2 0.1
T = 1 M Ave 100 100.2 99.8 99.9 100.1 99.9 100.0 99.9 T = 1 M % 0.6
0.3 0.4 0.3 0.1 0.1 0.1 0.1 RSD T = 2 M Ave 100.2 100.5 100.1 99.1
100.1 100.0 100.0 100.0 T = 2 M % 0.2 0.2 0.3 2.4 0.1 0.1 0.1 0.1
RSD T = 3 M Ave 100.0 100.0 99.9 99.5 100.1 100.1 100.1 100.0 T = 3
M % 3.4 0.4 0.5 0.5 0.2 0.1 0.1 0.0 RSD T = 4.5 M Ave 100.0 100.1
99.9 100.0 100.0 100.0 100.0 99.9 T = 4.5 M % 0.4 0.3 0.3 0.4 0.1
0.1 0.1 0.1 RSD T = 6 M Ave 98.7 99.1 99.2 99.2 99.6 99.8 99.5 99.6
T = 6 M % 0.8 0.6 0.6 0.4 0.3 0.1 0.1 0.3 RSD
[0256] The tests above were used to establish the stability of egg
white powder capsules for the exemplary formulations. The loss on
Drying results in Table 65, for 0.2, 1, 10, and 100 mg, show that
the loss on drying for the formulations varied a small amount, but
there was no significant difference between 1 month, 2 months, 3
months, 4.5 months and 6 months.
[0257] Ovomucoid was used as an exemplary egg protein to test the
specific potency of the protein over time. The potency of Ovomucoid
(ELISA) presented in Table 66 again showed some variation, but no
consistent loss with time up to 6 months. The HPLC assay results
presented in Table 67 show no significant loss in egg proteins over
the 6 months. Content Uniformity (Table 68), blend Uniformity
(Table 69), and deliverable Mass data (Table 70) confirmed these
results.
[0258] The results at 6 months show that at both temperatures in
this formulation the protein moisture content and physical
characteristics are unchanged. Thus, the tested blends were stable
for 6 months and, therefore, pharmaceutical preparations of the
formulations can be used reliably for at least 6 months.
Example 10
Eleven Month Stability Study
[0259] The stability of egg white protein capsules was analyzed for
11 months for 0.2 mg, 1.0 mg, 10 mg, and 100 mg capsules. The
stability protocol in Example 9 was used. All blends for this study
were processed as GMP demonstration batches using Michael Foods egg
white protein powder (lot 4043W-3). The capsules used for this
study were produced in the following batches: D14089, 0.2 mg egg
white protein capsules; D14084, 1.0 mg egg white protein capsules;
D14072, 10.0 mg egg white protein capsules; and D14070, 100.0 mg
egg white protein capsules.
TABLE-US-00071 TABLE 71 Physical Characteristics of Batch 14089
(0.2 mg) Time point Appearance Odor T = 0 white capsule containing
white, free-flowing no distinct odor fine powder with few clumps T
= 1 M white capsule containing white, free-flowing no distinct odor
fine powder with few clumps T = 2 M white capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 3 M
white capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 4.5 M white capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 6 M
white capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 11 M white capsule containing white,
free-flowing no distinct odor fine powder with few clumps
TABLE-US-00072 TABLE 72 Physical Characteristics of Batch 14084
(1.0 mg) Time point Appearance Odor T = 0 clear capsule containing
white, free-flowing no distinct odor fine powder with few clumps T
= 1 M clear capsule containing white, free-flowing no distinct odor
fine powder with few clumps T = 2 M clear capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 3 M
clear capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 4.5 M clear capsule containing white,
free-flowing no distinct odor fine powder with few clumps T = 6 M
clear capsule containing white, free-flowing no distinct odor fine
powder with few clumps T = 11 M clear capsule containing white,
free-flowing no distinct odor fine powder with few clumps
TABLE-US-00073 TABLE 73 Physical Characteristics of Batch 14072
(10.0 mg) Time point Appearance Odor T = 0 dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
1 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor T = 2 M dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
3 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor T = 4.5 M dark blue capsule; white
free-flowing fine faint acetic powder with few clumps acid odor T =
6 M dark blue capsule; white free-flowing fine faint acetic powder
with few clumps acid odor T = 11 M dark blue capsule; white
free-flowing fine faint acetic (25.degree. C. powder acid odor 60%
RH) T = 11 M dark blue capsule; white free-flowing fine faint
acetic (30.degree. C. powder acid odor 60% RH)
TABLE-US-00074 TABLE 74 Physical Characteristics of Batch 14070
(100.0 mg) Time point Appearance Odor T = 0 Burnt orange capsule
with gray bar on cap, black faint bar on body; off-white,
free-flowing fine powder acetic with few clumps acid odor T = 1 M
Burnt orange capsule with gray bar on cap, black faint bar on body;
off-white, free-flowing fine powder acetic with few clumps acid
odor T = 2 M Burnt orange capsule with gray bar on cap, black faint
bar on body; off-white, free-flowing fine powder acetic with few
clumps acid odor T = 3 M Burnt orange capsule with gray bar on cap,
black faint bar on body; off-white, free-flowing fine powder acetic
with few clumps acid odor T = 4.5 M Burnt orange capsule with gray
bar on cap, black faint bar on body; off-white, free-flowing fine
powder acetic with few clumps acid odor T = 6 M Burnt orange
capsule with gray bar on cap, black faint bar on body; off-white,
free-flowing fine powder acetic with few clumps acid odor T = 11 M
Burnt orange capsule with gray bar on cap, black faint bar on body;
off-white, free-flowing fine powder acetic acid odor
TABLE-US-00075 TABLE 75 Loss on Drying Batch Batch Batch Batch
D14089 D14084 D14072 D14070 (0.2 mg) (1 mg) (10 mg) (100 mg) Time T
= 0, 2.7% T = 0, 3.9% T = 0, 4.4% T = 0, 5.2% Condition 25/60 30/65
25/60 30/65 25/60 30/65 25/60 30/65 T = 1 M 3.7% 3.5% 5.1% 4.2%
5.3% 4.7% 5.8% 5.4% T = 2 M 3.5% 3.1% 4.2% 4.3% 4.6% 4.5% 5.5% 5.1%
T = 3 M 3.1% 3.3% 4.3% 4.3% 4.5% 4.5% 5.5% 5.2% T = 4.5 M 3.1% 3.5%
4.2% 4.4% 4.5% 4.5% 5.3% 5.3% T = 6 M 4.7% 4.1% 5.1% 4.9% 5.0% 4.7%
5.8% 5.6% T = 11 M 3.7% 4.3% 4.5% 5.0% 4.6% 4.7% 5.5% 5.6%
TABLE-US-00076 TABLE 76 Deliverable Mass (Reported as Percent)
Batch Batch Batch Batch D14089 D14084 D14072 D14070 Time (0.2 mg)
(1 mg) (10 mg) (100 mg) Condition 25/60 30/65 25/60 30/65 25/60
30/65 25/60 30/65 T = 0 Ave 100.5 100.2 100.1 100.1 % RSD 0.4 0.3
0.2 0.1 T = 1 M Ave 100 100.2 99.8 99.9 100.1 99.9 100.0 99.9 % RSD
0.6 0.3 0.4 0.3 0.1 0.1 0.1 0.1 T = 2 M Ave 100.2 100.5 100.1 99.1
100.1 100.0 100.0 100.0 % RSD 0.2 0.2 0.3 2.4 0.1 0.1 0.1 0.1 T = 3
M Ave 100.0 100.0 99.9 99.5 100.1 100.1 100.1 100.0 % RSD 3.4 0.4
0.5 0.5 0.2 0.1 0.1 0.0 T = Ave 100.0 100.1 99.9 100.0 100.0 100.0
100.0 99.9 4.5 M % RSD 0.4 0.3 0.3 0.4 0.1 0.1 0.1 0.1 T = 6 M Ave
98.7 99.1 99.2 99.2 99.6 99.8 99.5 99.6 % RSD 0.8 0.6 0.6 0.4 0.3
0.1 0.1 0.3 T = Ave 100.2 100.1 100.0 99.8 100.0 100.0 99.9 99.9 11
M % RSD 0.2 0.5 0.2 0.8 0.1 0.1 0.1 0.1
[0260] Formulations for the batches are given in Tables 57 through
60 above. Appearance and Odor are summarized in Tables 71-74. Loss
on Drying results are presented in Table 75. Deliverable Mass
results are provided in Table 76. Tables 77 and 78 provide data
about the egg protein after 11 months, showing that at 11 months
the HPLC assay results (Table 77) are not significantly different
from those at 1 month, 2 months, etc.
TABLE-US-00077 TABLE 77 Assay (HPLC) Batch Batch Batch Batch D14089
D14084 D14072 D14070 Time (0.2 mg) (1 mg) (10 mg) (100 mg)
Condition 25/60 30/65 25/60 30/65 25/60 30/65 25/60 30/65 T = 0
95.5 100.0 102.4 110.0 T = 1 M 113.5 113.8 97.5 96.6 97.4 96.3
106.3 107.2 T = 2 M 87.9 85.9 96.6 94.9 97.9 96.2 104.3 101.8 T = 3
M 86.0 79.9 96.7 92.5 100.5 98.0 102.8 105.0 T = 4.5 M 83.5 78.8
88.3 89.3 93.3 89.3 97.5 99.6 T = 6 M 87.7 80.1 91.1 85.8 94.9 89.8
102.7 100.8 T = 11 M 82.2 71.6 90.3 80.4 92.0 87.3 118.2 117.9
TABLE-US-00078 TABLE 78 Potency of Ovomucoid (ELISA) Batch Batch
Batch Batch D14089 D14084 D14072 D14070 Time (0.2 mg) (1 mg) (10
mg) (100 mg) Condition 25/60 30/65 25/60 30/65 25/60 30/65 25/60
30/65 T = 0 Data not available at T = 0; Method Development was
still in progress. T = 1 M 91.1 85.4 89.3 73.8 96.2 107.2 89.9
115.2 T = 2 M 41.3 42.2 49.9 51.5 81.8 96.3 100.1 106.1 T = 3 M
65.0 69.2 67.3 93.9 71.8 91.2 94.5 85.4 T = 4.5 M 69.3 76.6 88.9
81.3 90.2 94.8 90.3 96.8 T = 6 M 99* 83* 90** 93** 86** 84** 91**
73** 75** 63** T = 11 M 100* 105* 111* 121* 76** 76** 78** 85**
84** 85** 93** 102** *With placebo spiked standard **With regular
standard
[0261] The results of the 11 month stability study show that the
dosage, moisture content, total protein and ovomucoid potency stays
the same within acceptable variation. Therefore, the formulation
provides an environment that allows the moisture content to stay
stable. However, Table 78 shows that there was some loss in potency
at high temperature, suggesting the formulations should be kept no
higher than at room temperature for best stability.
Example 11
Manufacturing Protocol
[0262] The following materials were screened through a 20-mesh
screen and added to a 16 quart V-blender: Dried Egg White protein
and ProSolv.RTM. SMCC50 (Silicified Microcrystalline Cellulose,
NF). The material was blended for 5 minutes. The contents of the 16
quart V-shell were discharged and screened through a 60-mesh
screen. The screened material was returned to the 16 quart V-shell.
Rinse the empty bag with ProSolv.RTM. SMCC50 and screen the rinse
material through a 60 mesh, return the screened material to the 16
quart V-shell and blend for 10 minutes. Repeat.
[0263] Screen the following materials in order through a 20 mesh
screen and add to the 16 quart V-blender: ProSolv.RTM. SMCC50.
Blend for 15 minutes. Screen the following materials in order
through a 20-mesh screen and add to the 16 quart V-blender:
ProSolv.RTM. SMCC HD90 and Mannitol, NF (Pearlitol.RTM. 200SD).
Blend the combined material for 15 minutes. Pass Magnesium
Stearate, NF (Hyqual.RTM. Vegetable Source) through a 40 mesh
screen and add to the blend. Blend for 10 minutes. Discharge the
combined blend into an appropriately sized container lined with 2
polyethylene bags. Analyze the protein content and uniformity of
the blend and package appropriately into a capsule or pouch.
VII. Conclusion
[0264] Unless the context clearly requires otherwise, throughout
the description and the claims, the words `comprise`, `comprising`,
and the like are to be construed in an inclusive sense as opposed
to an exclusive or exhaustive sense; that is to say, in the sense
of "including, but not limited to". Words using the singular or
plural number also include the plural or singular number,
respectively. Additionally, the words "herein," "above" and "below"
and words of similar import, when used in this application, shall
refer to this application as a whole and not to any particular
portions of this application.
[0265] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0266] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while process steps, formulation components
or functions are presented in a given order, alternative
embodiments may include these in a different order, or
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other compositions, not only the
compositions described herein. The various embodiments described
herein can be combined to provide further embodiments.
[0267] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while aspects associated with certain embodiments of
the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such aspects, and
not all embodiments need necessarily exhibit such aspects to fall
within the scope of the disclosure. Accordingly, the disclosure is
not limited, except as by the appended claims.
* * * * *