U.S. patent application number 09/978112 was filed with the patent office on 2002-03-21 for delivery system to modulate immune response.
This patent application is currently assigned to ALLERGENICS, INC.. Invention is credited to Rivera, Roberto L..
Application Number | 20020034514 09/978112 |
Document ID | / |
Family ID | 21916921 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034514 |
Kind Code |
A1 |
Rivera, Roberto L. |
March 21, 2002 |
Delivery system to modulate immune response
Abstract
A microsphere containing an immunogen bound to an inert particle
having a mesh size of greater than about 35 mesh for site-specific
release and induction of an immune response. The immune response
may be an overall enhanced T lymphocyte immune response or a
selective response. The physical and chemical characteristics
and/or modes of administration of the microsphere may be engineered
to increase T.sub.H1 lymphocytes for treatment of cancer or
infectious disease. The microencapsulated immunogen has an enteric
coating for oral administration.
Inventors: |
Rivera, Roberto L.;
(Cincinnati, OH) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
ALLERGENICS, INC.
395 OYSTER POINT BOULEVARD, SUITE 113
SOUTH SAN FRANCISCO
CA
|
Family ID: |
21916921 |
Appl. No.: |
09/978112 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09978112 |
Oct 15, 2001 |
|
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09041514 |
Mar 12, 1998 |
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Current U.S.
Class: |
424/184.1 ;
424/493 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 9/5078 20130101; A61P 35/00 20180101; A61P 31/00 20180101 |
Class at
Publication: |
424/184.1 ;
424/493 |
International
Class: |
A61K 039/00; A61K
009/50 |
Claims
What is claimed is:
1. A method of inducing an immune response in a mammal comprising
administering a microsphere comprising an immunogen bound to an
inert particle to a small intestine of said mammal, said inert
particle having a mesh size greater than about 35 mesh.
2. The method of claim 1 wherein said microsphere is administered
orally and said microsphere comprises an enteric coated
microsphere.
3. The method of claim 1 wherein said microsphere is administered
in a gel capsule.
4. The method of claim 1 wherein said immunogen is selected from
the group consisting of a peptide, a protein fragment, a protein, a
gene, a gene fragment, a DNA, an RNA and combinations thereof.
5. The method of claim 1 wherein said immunogen is a vaccine.
6. The method of claim 1 further comprising administering a
potentiating agent bound to an inert particle, said inert particle
selected from the group consisting of an immunogen-bound inert
particle and a non-immunogen bound inert particle.
7. The method of claim 1 wherein a plurality of microspheres are
administered to selectively induce the immune response.
8. The method of claim 7 wherein said microspheres have
compositions selected from the group consisting of different inert
particle sizes, different inert particle compositions, different
enteric coatings, different formulations and combinations
thereof.
9. The method of claim 1 wherein said microsphere containing said
immunogen induces an increase in the number of T lymphocytes.
10. The method of claim 9 wherein said microsphere containing said
immunogen induces an increase in a cell population selected from
the group consisting of a T.sub.H1 lymphocyte, a cytotoxic T
lymphocyte (CTL), and combinations thereof.
11. The method of claim 1 wherein said inert particle has a mesh
size greater than about 40 mesh.
12. The method of claim 1 where said immunogen is contained on an
inert particle selected from the group consisting of a nonpareil, a
silica powder, a salt crystal and a sugar crystal.
13. A method of treating cancer in a mammal comprising
administering a microsphere comprising an immunogen bound to an
inert particle to a small intestine of said mammal, said inert
particle having a mesh size greater than about 35 mesh.
14. The method of claim 13 wherein said microsphere is administered
orally and said microsphere comprises an enteric coated
microsphere.
15. The method of claim 13 wherein said microsphere is administered
in a gel capsule.
16. The method of claim 13 wherein said immunogen is selected from
the group consisting of a peptide, a protein fragment, a protein, a
gene, a gene fragment, a DNA, an RNA and combinations thereof.
17. The method of claim 13 wherein said immunogen is a vaccine.
18. The method of claim 13 further comprising administering a
potentiating agent bound to an inert particle, said inert particle
selected from the group consisting of an immunogen-bound inert
particle and a non-immunogen bound inert particle.
19. The method of claim 13 wherein a plurality of microspheres are
administered to selectively induce the immune response.
20. The method of claim 19 wherein said microspheres have
compositions selected from the group consisting of different inert
particle sizes, different inert particle compositions, different
enteric coatings, different formulations and combinations
thereof.
21. The method of claim 13 wherein said microsphere containing said
immunogen induces an increase in the number of T lymphocytes.
22. The method of claim 21 wherein said microsphere containing said
immunogen induces an increase in a cell population selected from
the group consisting of a T.sub.H1 lymphocyte, a cytotoxic T
lymphocyte (CTL), and combinations thereof.
23. The method of claim 13 wherein said inert particle has a mesh
size greater than about 40 mesh.
24. The method of claim 13 wherein said immunogen is contained on
an inert particle selected from the group consisting of a
nonpareil, a silica powder, a salt crystal and a sugar crystal.
25. A method of inducing an immune response in a mammal comprising
orally administering to said mammal a microsphere comprising an
enteric-coated inert particle containing a protein immunogen, said
particle having a mesh size greater than about 40 mesh.
26. The method of claim 25 further comprising administering a
potentiating agent bound to an inert particle, said inert particle
selected from the group consisting of an immunogen-bound inert
particle and a non-immunogen bound inert particle.
27. The method of claim 25 wherein a plurality of microspheres are
administered to selectively induce the immune response.
28. The method of claim 27 wherein said microspheres have
compositions selected from the group consisting of different inert
particle sizes, different inert particle compositions, different
enteric coatings, different formulations and combinations
thereof.
29. The method of claim 25 wherein the immune response comprises an
increase in a T lymphocyte population.
30. The method of claim 29 wherein the immune response comprises an
increase in a cell population selected from the group consisting of
a T.sub.H1 lymphocyte, a cytotoxic T lymphocyte (CTL), and
combinations thereof.
31. A composition adapted to induce an immune response comprising
an immunogen contained on an inert particle and having an enteric
coating, said inert particle having a mesh size greater than about
35 mesh.
32. The composition of claim 31 contained in a gel capsule.
33. The composition of claim 31 further comprising a potentiating
agent.
Description
FIELD OF THE INVENTION
[0001] This invention is directed generally to a method of
selecting and/or selectively modulating an immune response by
administering a microencapsulated immunogen.
BACKGROUND OF THE INVENTION
[0002] The immune system recognizes and distinguishes substances as
self versus nonself, and defends the body against nonself
substances. The importance of this distinction is evident in a
variety of conditions such as autoimmune diseases, rejection of
transplanted tissues or organs, allergic reactions, cancer and
infectious diseases, and modes of treatments such as immunotherapy
and gene therapy. For example, in autoimmune diseases such as
rheumatoid arthritis, systemic lupus erythematosus and myasthenia
gravis, the body mistakenly treats self as nonself and thus
destroys its own components. In transplant rejection,
immunosuppressive drugs are administered to a recipient to prevent
the recipient's immune system from rejecting a true nonself
substance so that the recipient can accept the transplanted tissue
or organ as its own. In allergic reactions such as asthma, eczema
and hay fever, there is an immune hypersensitivity in some
individuals that occurs immediately following contact with an
antigen. In infectious diseases a microbe such as a bacterium,
parasite or virus stimulates an immune response. The microbe or a
microbe subunit may be formulated as a vaccine to provide
prophylactic protection against subsequent infection. In cancer,
unlike the other conditions, an immune response is not mounted and
the lack of an immune response plays a role in the uncontrolled
growth of malignant cells. A wide variety of foreign substances,
termed antigens or immunogens, elicit an immune response and thus
are targeted by the immune system. Examples of antigens include,
but are not limited to, infectious disease agents such as bacteria,
viruses, parasites and fungi as well as mites, pollen, animal
dander, drugs, toxins and chemicals.
[0003] The immune system is a complex network of cells, tissues and
organs that directly and indirectly target and ultimately destroy
foreign substances. Of the various cells involved in mounting an
immune response, lymphocytes are one type of white blood cells that
have a crucial role. One type of lymphocyte is the B lymphocyte (B
cell) that targets and indirectly destroys foreign substances by
mounting a humoral immune response to produce antibodies against
specific antigens. The other type of lymphocyte is the T lymphocyte
(T cell) that targets and directly kills foreign substances by
mounting a cell-mediated immune response. There are three major
subtypes of T cells designated as T helper cells, T suppressor
cells, and T cytotoxic cells. T helper cells are of two types:
T.sub.H1 and T.sub.H2 cells. T.sub.H2 cells help B cells mount a
humoral immune response and help T cytotoxic cells maintain
themselves by producing growth factors needed by the T cytotoxic
cells. T.sub.H2 cells express the CD4 glycoprotein antigen. T
suppressor cells inhibit or suppress T helper cells; they express
the CD8 glycoprotein antigen. T cytotoxic cells, also called
cytotoxic T lymphocytes (CTL), express the CD8 glycoprotein antigen
and are a subset of T cells that kill cells expressing a specific
antigen upon direct contact with these target cells. Pre-CTL are T
cells that are committed to the CTL lineage, have undergone thymic
maturation and are already specific for a particular antigen, but
lack cytolytic function. CTL are important effector cells in three
settings: (1) intracellular infections of non-phagocytic cells or
infections that are not completely contained by phagocytosis such
as viral infections, (2) infections by bacteria such as Listeria
monocytogenes, and (3) acute allograft rejection and rejection of
tumors.
[0004] An immunogenic response is most predictably induced by using
a protein as the immunogen. In immunotherapy, the protein is
frequently administered parenterally, for example by injection.
While injections are inconvenient and uncomfortable to many
patients, they have heretofore been a common route of
administration because orally administered protein is degraded by
protease enzymes and acid in the stomach and enzymes in the small
intestines. It has been demonstrated that oral administration of a
soluble protein such as the model antigen ovalbumin (OVA) results
in the induction of immune tolerance, characterized by the loss of
either antibody or T cell response to the protein antigen. However,
U.S. Pat. No. 5,591,433 discloses that immunologically active
biomolecules and other therapeutic proteins can be orally
administered by microencapsulating the protein and coating the
microsphere to form a pH-sensitive enterocoated microsphere
particle that is resistant to the action of digestive proteolytic
enzymes and acids. The microspheres disclosed in the '433 patent
consist of protein bound to an inert particle having a mesh size of
about 30-35 mesh (about 600 .mu.m to about 500 .mu.m) diameter and
coated with an acid stable polymer. What is needed, however, is a
method of better selecting and selectively modulating a particular
immune response from the complex immune repertoire to better
respond to different antigenic stimuli in different conditions
requiring treatment.
[0005] For example, current cancer treatments include combinations
of chemotherapy, radiation therapy, and surgical excision of some
or all of a solid tumor. Each of these treatment mechanisms is
targeted to eliminating malignant cells but is performed at the
expense of destroying nonmalignant cells. Thus, none of these
treatments utilize the body's own capacity for cell destruction,
namely, the immune system and particularly the cytotoxic T cells,
to kill malignant cells. A method of increasing an immune response
and/or selectively stimulating the cytotoxic T cell population
would therefore be a valuable supplement to traditional treatment
methods. In addition, such a method would operate without the
adverse effects of chemotherapeutic drugs, radiation, or surgical
insult. Cancer cells, however, are not recognized as foreign by the
immune system and thus are not targeted for destruction. One goal
in developing cancer treatments is to stimulate the immune system
to mount an immune response against cancer cells. Of the three
major T cell types, the T cytotoxic cells frequently directly
target and destroy cancer cells. Thus, selectively increasing the T
cytotoxic cell subtype may be an advantageous way to check the
unregulated cell division that is a hallmark of cancer cells.
[0006] As another example, the T cytotoxic cells also directly
target and destroy extracellular infectious disease agents and
infectious disease agents in infected cells. Cell mediated immunity
consists of two types of reactions.
[0007] The first type is macrophage activation resulting in the
killing of phagocytized microbes. The second type is lysis of
infected cells by CD8+ cytotoxic T lymphocytes (CTL). Differences
among individuals in the patterns of immune responses to
intracellular microbes, for example in HIV infection, are important
determinants of disease progression and clinical outcome. The
selective increase in the T cytotoxic cell subtype may be used to
combat infectious diseases.
[0008] There is thus a need for a method and composition to better
modulate and/or selectively stimulate an immune response. Such a
method and composition would find wide use in immunotherapy or gene
therapy for conditions such as allergies, infectious diseases,
cancer, transplant rejection, and autoimmune diseases. Such a
method and composition would also be a valuable prophylactic and/or
therapeutic supplement to current methods of treating these
conditions.
SUMMARY OF THE INVENTION
[0009] This invention provides methods and compositions to induce
an enhanced general or selective immune response. A drug delivery
system comprises a microsphere of an immunogen bound to an inert
particle having a mesh size greater than about 35 mesh. The
microsphere is administered to the small intestine of a mammal. The
microsphere is preferably administered orally and contains one or
more enteric coatings and may be administered in a gel capsule. In
one embodiment the inert particle has a mesh size greater than
about 40 mesh and may be a nonpareil, a silica powder, a salt
crystal or a sugar crystal.
[0010] The response may encompass a general enhanced production of
T.sub.H1 cells, T.sub.H2 cells and cytotoxic T lymphocyte (CTL)
subsets, or an enhanced shift from a T.sub.H2 type response to a
T.sub.H1 type response, or an enhanced shift from a T.sub.H1 type
response to a T.sub.H2 type response, or an enhanced
differentiation of pre-CTL to CTL. The immunogen may be a peptide,
a protein fragment, a protein, a DNA, and/or an RNA, and may be a
gene, a gene fragment or a vaccine.
[0011] The immunogen may be administered in a dosing regimen and/or
a dosing composition containing a number of microspheres to
selectively induce a particular immune response. The microspheres
of the dose may contain the same enteric coatings or different
enteric coatings, the same formulation or different formulations,
and/or the same inert particle core composition and size or
different core compositions and sizes. The immunogen may also be
administered with a potentiating agent, either in a single inert
particle or in separate inert particles. If formulated with the
immunogen and potentiating agent in a single inert particle, the
various single inert particles of the administered dose may have
the same enteric coating or a different enteric coating, the same
formulations or different formulations, and/or the same inert
particle core composition and size or different core compositions
and sizes. Likewise, if formulated with the immunogen and
potentiating agent in separate inert particles, the separate
microspheres of the administered dose may have the same enteric
coatings or different enteric coatings, the same formulations or
different formulations, and/or the same inert core compositions and
sizes or different core compositions and sizes.
[0012] As will be appreciated, the disclosed delivery system and
methods of using the system have a wide array of applications.
These and other advantages of the invention will be further
understood with reference to the following drawings, detailed
description and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph of the results of primary lymphocyte
proliferation with different modes of ovalbumin (OVA)
administration.
[0014] FIG. 2A is a graph of the results of a lymphoproliferative
analysis using either microspheres containing OVA, OVA in adjuvant,
or placebo microspheres, and
[0015] FIG. 2B is a graph of the results using concanavalin A (Con
A) nonspecific mitogen stimulation.
[0016] FIG. 3A is a graph of the results from in vitro stimulation
with microspheres containing OVA, and
[0017] FIG. 3B is a graph of the results using Con A nonspecific
mitogen stimulation.
[0018] FIG. 4 is a graph of cytotoxic T lymphocyte responses at
different effector:target ratios.
[0019] FIG. 5A is a graph of the results of antibody blocking
experiments for microspheres containing OVA, and
[0020] FIG. 5B is a graph of the results using Con A nonspecific
mitogen stimulation.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Definition of Terms
[0022] The terms immunogen or antigen are broadly used herein to
encompass any chemical or biological substance that elicits an
immune response when administered to a mammal. While an immunogen
is frequently a protein, it may also be a nucleic acid. For the
purpose of the present invention, immunogens include but are not
limited to the following: allergenic proteins and digested
fragments thereof such as pollen allergens from ragweed, rye, June
grass, orchard grass, sweet vernal grass, red top grass, 15 timothy
grass, yellow dock, wheat, corn, sagebrush, blue grass, California
annual grass, pigweed, Bermuda grass, Russian thistle, mountain
cedar, oak, box elder, sycamore, maple, elm and so on, dust, mites,
bee and other insect venoms, food allergens, animal dander,
microbial vaccines which in turn include viral, bacterial,
protozoal, nematode and helminthic vaccines and their various
components such as surface antigens, including vaccines which
contain glycoproteins or proteins, protein fragments, genes or gene
fragments prepared from, for example, Staphylococcus aureus,
Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria
meningitidis, Neisseria gonorrhoeae, Salmonellae species, Shigellae
species, Escherichia coil, Klebsiellae species, Proteus species,
Vibrio cholerae, Helicobacter pylori, Pseudomonas aeruginosa,
Haemophilus influenzae, Bordetella pertussis, Mycobacterium
tuberculosis, Legionella pneumophila, Treponema pallidum, and
Chlamydiae species, tetanus toxoid, diphtheria toxoid, influenza
viruses, adenoviruses, paramyxoviruses, rubella viruses,
polioviruses, hepatitis viruses, herpesviruses, rabies viruses,
human immunodeficiency viruses, and papilloma viruses, in addition
to protozoal parasites such as Toxoplasma gondii, Pneumocystis
carinii, Giardia lamblia, Trichomonas vaginalis, Isospora beeli,
Balantidium coli, Blastocystis hominis, and the various species of
Entamoeba, Amebae, Plasmodium, Leishmania, Trypanosoma, Babesia,
Cryptosporidium, Sarcocystis, and Cyclospora, as well as nematodes
and helminths of the various species of trematodes, flukes,
cestodes and visceral larvae.
[0023] Immunogens may be administered as therapeutic or
prophylactic agents, either with or without a potentiating agent. A
therapeutic immunogen is defined herein as one that alleviates a
pathological condition or disease. Therapeutic agents that may be
used in the present invention include, but are not limited to,
immunogenic agents and gene therapy agents. A prophylactic agent is
defined herein as one that either prevents or decreases the
severity of a subsequently acquired disease or pathological
process. An example of a prophylactic agent is a vaccine against a
microbe causing an infectious disease. A potentiating agent is
defined herein as one that enhances the antigenicity of other
immunogens. A potentiating agent thus indirectly stimulates an
immune response. An example of a potentiating agent is an adjuvant,
defined herein as any biological or chemical substance which, when
administered with an immunogen, enhances the immune response
against the immunogen. Examples of adjuvants are inorganic salts
such as aluminum hydroxide (alum), cytokines, and bacterial
endotoxins such as cholera toxin B (CTB). Another example of a
potentiating agent is a hapten, defined herein as a low molecular
weight substance that itself is non-immunogenic but becomes
immunogenic when conjugated to a high molecular weight carrier.
Other potentiating agents include bioadhesives, mucoadhesives and
promoting agents.
[0024] Microsphere Formulations
[0025] As used herein and unless specifically indicated otherwise,
all percentages are given in terms of the weight of the ingredient
relative to the total weight of the microsphere. In one embodiment
of the invention, an aqueous solution of the immunogen with an
optional stabilizing agent to provide physical protection for the
immunogen is formed. The aqueous immunogen solution will generally
be from about 0.5% to about 10% by weight of the immunogen in the
microsphere, with about 1% being preferred.
[0026] Stabilizing agents are generally therapeutically inactive,
water soluble sugars that act to protect the immunogen during a
step in the formulation of the immunogen and/or during a subsequent
coating step. Examples of stabilizing agents include the sugars
lactose, mannitol and trehalose. The stabilizing agent is added at
a concentration of from about 0.1% to about 10%, with a
concentration of about 5% being preferred. If the immunogen
solution has a low viscosity, it may be desirable to add from about
1% to about 10% of polyvinyl pyrrolidone or other binding agents
such as hydroxypropylcellulose or hydroxypropylmethylcellulose to
bind the immunogen to the inert particle.
[0027] The solution of one or more immunogens and an optional
stabilizing agent is then applied, for example by spraying, to a
pharmaceutically inert material substrate, hereinafter termed an
inert particle. The inert particle may encompass a variety of
shapes and forms such as a bead, a sphere, a powder, a crystal, or
a granule. In one embodiment, a nonpareil, defined as a small round
particle of a pharmaceutically inert material, may be used. One
such nonpareil is available under the brand name NuPareils.RTM.
(Crompton & Knowles Corp., Mahwah, N.J.). In other embodiments,
a silica powder, sugar crystal or salt crystal may be used. The
inert particle in whatever shape or form has a mesh size greater
than about 35 mesh, preferably greater than about 40 mesh, and most
preferably in the range of about 45 to 200 mesh.
[0028] Glatt.RTM. brand powder coater granulators such as the
GPCG-1 HS, GPCG-5HS, or GPCG-60HS fluid bed coaters are suitable
for use to coat the immunogen onto the inert particle. Various
other brands of Wurster type fluid bed coaters (NIRO, Vector, Fluid
Air, etc.) are also suitable for use. Coating conditions and times
vary depending on the apparatus and coating viscosity; however,
coating must generally be conducted at temperatures less than about
50.degree. C., and preferably less than about 35.degree. C., to
avoid denaturation of a protein immunogen.
[0029] The dry immunogen-coated inert particles are preferably also
coated with one or more layers of acid stable polymers to form an
enteric coating. This coating renders the immunogen resistant to
degradation in the acid environment of the stomach. In addition,
varying the composition and/or amount of the enteric coating may
allow the enteric coating to dissolve, and thus release the
immunogen, at a particular pH in the small intestine for an
optimally selective T cell response. The coating of one or more
polymers may be applied in a similar manner and with similar
equipment as the coating steps previously described.
[0030] The enteric coating is preferably a water-based emulsion
polymer such as ethylacrylate methacrylic acid copolymer, sold as
Eudragit.RTM. L-30D (Huls America Inc., Somerset, N.J.) with a
molecular weight of about 250,000 and generally applied as a
30%.sup.wv aqueous dispersion. Some examples of alternative polymer
coatings are the solvent free Eudragit L/S 100 or
hydroxypropylmethyl cellulose acetate succinate. The enteric
coating allows the microencapsulated immunogen to be orally
administered without being released from the microsphere until
encountering a specific region of the gut. The chemical composition
of the enteric coating may be formulated to dissolve, and thus
release the immunogen, at a particular pH in the small intestine
for an optimally selective T cell response. Alternatively, the
enteric coating may be formulated to release the immunogen after
encountering sufficient mechanical and/or chemical erosion.
[0031] The coating composition may be combined with a plasticizer
to improve the continuity of the coating. Several well known
plasticizers may be used, with triethylcitrate (Morflex Inc.,
Greensboro, N.C.) preferred. Although plasticizers can be liquid,
they are not considered to be solvents since they lodge within the
coating and alter its physical characteristics but do not act to
dissolve the protein immunogen. A plasticizer which dissolves or
denatures the immunogen would be unacceptable.
[0032] Talc (about 3.0%) may be added to prevent the particles from
sticking to each other. An antifoaming agent (about 0.0025%) such
as sorbitan sesquioleate (Nikko Chemicals Co. Ltd., Japan) or
silicone can also be added. An antistatic agent (about 0.1%) such
as Syloid 74FP (Davison Chemical Division, Cincinnati, Ohio) can be
added. The talc, antifoaming agent and antistatic agent are added
only if needed.
[0033] The inert particles containing the immunogen, the optional
stabilizing agent or agents and other formulation ingredients are
dried and may be coated with the enteric coating as previously
described. The coating solution is about 30% to about 75% polymer,
about 0% to about 10% plasticizer, about 0% to about 3% talc, about
0% to about 0.0025% antifoaming agent, about 0% to 3% antistatic
agent and water. It is generally preferable that there be no
organic solvents, including alcohols and even glycols, present in
the coating composition as organic solvents can denature the
immunogen.
[0034] Potentiating Agents
[0035] In an alternative embodiment, a potentiating agent may be
added to increase the immunogenicity of the protein. Examples of
potentiating agents include adjuvants, bioadhesives, mucoadhesives,
and promoting agents. Adjuvants work by either concentrating
antigen at a site where lymphocytes are exposed to the antigen or
by inducing cytokines which regulate lymphocyte function. The
adjuvant may be either a biological compound, a chemical compound
that is therapeutically acceptable, or a combination of a
biological and chemical compound. Examples of chemical adjuvants
are water dispersible inorganic salts such as aluminum sulfate,
aluminum hydroxide (alum) and aluminum phosphate. Examples of
biological adjuvants are endogenous cytokines such as
granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor
necrosis factor-a (TNF-.alpha.), interleukin-2 (IL-2),
interleukin-4 (IL-4), interleukin-12 (IL-12) and .gamma.-interferon
(IFN.gamma.), microorganisms such as BCG (bacille Calmette-Guerin),
Corynebacterium parvum, and Bordetella pertussis, bacterial
endotoxins such as cholera toxin B (CTB), lipopolysaccharide (LPS),
and muramyldipeptide (N-acetyl-muramyl -L-alanyl-D-isoglutamine
[MDP]). Commercially available adjuvants such as DETOX-PC.RTM. are
also available. Bioadhesives such as Lycopersicon esculentum lectin
(tomato lectin, LT) and mucoadhesives such as Chitosans-like
N-trimethyl chitosan chloride bind to sugars and form
glycoconjugates at site-specific areas of the intestines. Promoting
agents are defined herein as formulation ingredient(s) that promote
uptake, transport or presentation of antigen(s), adjuvants, or
haptens thereby enhancing the desired immune response. Examples of
promoting agents are glycoproteins, lipoproteins, bile salts, fatty
acids, phospholipids, glycolipids, triglycerides, and cholesterol,
cyclodextrins, glycerol, among others. All of the above
potentiating agents may be incorporated into the microsphere
formulation singly, in combination, or as part of covalent or
noncovalent complexes.
[0036] The potentiating agent may be added to the aqueous
dispersion or solution of immunogen prior to coating onto the inert
particle. Alternatively, the potentiating agent may be added to
non-immunogen bound inert particles. Generally, about 1% to about
10% of potentiating agent is added. The potentiating agent may be
bound to the same inert particle as the immunogen. Alternatively,
the potentiating agent may be bound to a first inert particle and
the immunogen may be bound to a second inert particle, such that
the potentiating agent may be applied to non-immunogen bound inert
particles.
[0037] Proposed Mechanism of Action
[0038] It has been found that microspheres produced from inert
particles having a mesh size greater than about 35 mesh enhance and
selectively stimulate T cytotoxic cells over other types of T
cells. As shown in FIG. 1, the microspheres of the present
invention have a potentiating agent-like effect and the extent of T
cell stimulation increases with decreasing size of the inert
particle of the microsphere. Microspheres containing OVA with an
inert particle mesh size greater than about 35 mesh (open bars)
stimulated primary lymphocytes more than twice as much as
microspheres containing both OVA and adjuvant with an inert
particle mesh size less than about 35 mesh (solid bars).
Microspheres containing OVA with a mesh size greater than about 35
mesh stimulated primary lymphocytes more than three times as much
as parenterally administered OVA with adjuvant (hatched bars). This
demonstrates that by using enteric coated immunogens attached to an
inert particle having a mesh size greater than about 40 mesh, a
potentiating agent-like effect in selecting for a T cytotoxic cell
response is produced that is equivalent to the response produced
using OVA administered with DETOX-PC.RTM. adjuvant. Thus, adding an
adjuvant such as aluminum hydroxide (alum) or DETOX-PC.RTM. or
other potentiating agent(s) to the microsphere formulation in
certain cases may provide additional stimulation of a T cytotoxic
cell population, and may allow a lower initial dose of
immunostimulatory drug to generate an immune response equivalent to
that obtained with a higher dose of immunostimulatory drug.
[0039] While the exact mechanism for these selective stimulations
is unclear, one explanation may be that smaller enteric antigen
coated particles provide an increase in contact points between the
immunogen encapsulated therein and the appropriate immune cell
receptor systems lying along the mammalian intestinal tract,
particularly in the diffuse lymphatic tissue of Peyer's patches.
These smaller particles also contain more of certain formulation
ingredients on a per weight basis, some of which may enhance
antigen presentation and delivery. Other explanations, however, may
be possible.
[0040] Microsphere Dosing
[0041] In use, the microspheres of the present invention,
comprising immunogen-bound inert particles having a mesh size
greater than about 35 mesh and enteric coated with an optional
potentiating agent, are administered in a dosing schedule and
composition comprising various permutations of the above sizes and
compositions to modulate an immune response. The microspheres are
preferably administered orally such as by gavage or feeding, or may
be administered parenterally such as by subcutaneous injection.
Dosing may be consecutive or intermittent and at various times and
in various formulations. As used herein, formulations encompass
both the different percentage compositions and different
physicochemical compositions of the microspheres, such as size,
coatings, polymers, plasticizers, anti-stick agents, anti-foam
agents, antistatic agents, potentiating agent(s) and
excipients.
[0042] For example, an administered dose may contain a number of
single inert particles with each inert particle containing one or
more immunogens and, if added, the potentiating agent. If
formulated as a single inert particle, the various single
microspheres of the administered dose may have the same enteric
coating or different enteric coatings, the same formulation or
different formulations of polymers, plasticizers, binding agents,
anti-stick agents, anti-foam agents, antistatic agents,
potentiating agent(s) and excipients, and/or the same inert core
composition and size or different inert core compositions and
sizes. Alternatively, the dose may be formulated to contain a
combination of inert particles with one or more immunogens and, if
added, the potentiating agent(s) in separate inert particles. If
formulated with the immunogen and potentiating agent(s) in separate
inert particles, the separate microspheres of the administered dose
may have the same enteric coatings or different enteric coatings,
the same formulations or different formulations of polymers,
plasticizers, binding agents, anti-stick agents, anti-foam agents,
antistatic agents, potentiating agent(s) and excipients, and/or the
same inert core compositions and sizes or different inert core
compositions and sizes. These various combinations and permutations
of inert particle size, inert particle composition, enteric
coating, and formula composition help to achieve selective
distribution and presentation of the antigen along the gut upon
administration of the microspheres.
[0043] The microspheres may be placed in gel capsules for oral
administration to humans or other mammals. Dosage will depend on
the individual and the course of the therapy. For example, in
treatment using the microspheres of the invention containing
ragweed as the immunogen, the dosage would be about 0.03 to about
35 units in terms of a major allergenic protein, Amb-a-1,
administered daily. Dosage for allergens may be different from the
dosage used in immunotherapy by injection.
[0044] Applications
[0045] In use, the microspheres of the present invention containing
an enteric coated immunogen and an optional potentiating agent have
numerous applications. For example microspheres containing
glycoproteins, proteins, protein fragments, peptides, or gene
fragments from microorganisms, viruses or parasites would be a
valuable prophylactic and/or therapeutic supplement to the typical
antimicrobial, antiviral and antiparasitic agents administered to
treat infectious diseases. As another example, a peptide fragment
containing nondominant epitope(s) from the HER-2/neu oncogenic
"self-protein" can be used as the immunogen in the microspheres of
the invention to increase the efficacy of a cancer vaccine by
breaking tolerance against overexpressed tumor proteins. This use
would be especially valuable since HER-2/neu is a "self" protein
and thus does not generate an immune response. By using a peptide
containing nondominant epitope(s) rather than the whole protein as
reported by Disis et al. (J. Immunol., 1996:156, 3151-3158) in the
microspheres of the invention, a cancer vaccine eliciting a T
cytotoxic cell response targeting "self" tumor antigens would be
produced. As still another example, the immunogen may be an
allergen that increases a T.sub.H1 type response and hence increase
production of typical T.sub.H1 cytokines such as y-interferon
(IFN-.gamma.), tumor necrosis factor-.beta.(TNF-.beta.), and
interleukin-2 (IL-2) which, in turn, may decrease inflammation in
allergic conditions such as asthma.
[0046] The invention will be further appreciated in light of the
following examples.
EXAMPLE 1
[0047] Tumor Cell Lines
[0048] The EL4 thymoma cell line (TIB-39) was obtained from
American Type Culture Collection (ATCC, Rockville, Md.). The cells
were maintained in culture using RPMI 1640 medium supplemented with
10% fetal calf serum (FCS) (HyClone Laboratories, Logan, Utah), 15
mM HEPES buffer, 2 mM glutamine, 0.1 mM non-essential amino acids,
50 units/ml penicillin, 50 units/ml streptomycin, 1 mM sodium
pyruvate (Biofluids, Rockville, Md.), and 50 .mu.M
2-mercaptoethanol (Sigma, St. Louis, Mo.).
[0049] Antigens
[0050] Purified chicken egg ovalbumin (OVA) (grade V) was purchased
from Sigma (St. Louis, Mo.). The H-2Kb restricted peptide epitope
of OVA protein, OVA.sub.257-264 (SIINFEKL), was synthesized using
FMOC chemistry on an Applied Biosystems Model 432A peptide
synthesizer. The lyophilized product was resuspended in water at a
concentration of 2 mg/ml, sterile filtered and stored at
-70.degree. C. The peptide was determined by high performance
liquid chromatography to be greater than 90% pure.
[0051] OVA protein was coated onto inert particles and the antigen
was encapsulated using an aqueous enteric coating system containing
a biodegradable polymethacrylic acid copolymer (Eudragit L30D). The
inert particles were NuPareils.RTM. measuring about 45 mesh.
[0052] Immunization
[0053] Six- to eight-week-old C57BL/6 (H-2K.sup.b) female mice were
obtained from Taconic Farms (Germantown, N.Y.). These animals were
immunized either by subcutaneous injection with 30 .mu.g OVA
protein emulsified in DETOX-PC.RTM. adjuvant (RIBI ImmunoChem
Research, Hamilton, Mont.), or orally via intubation into the back
of the throat with microspheres containing 200 .mu.g OVA. Control
mice were orally fed a placebo microsphere. A series of three
immunizations was performed on days 0, 14, and 28. Animals were
euthanized three weeks following the final immunization.
[0054] Lymphoproliferation
[0055] Spleens were removed from immunized animals 21 days after
their third immunization and were mechanically dispersed through a
70 .mu.m nylon cell strainer (Falcon; Becton Dickinson, Franklin
Lakes, N.J.) to yield a single cell suspension. Dead cells and
erythrocytes were removed by centrifugation over a Ficoll-Hypaque
gradient (d=1.119 g/cm). The recovered cell population was then
enriched for T cells by passing the splenic mononuclear cells over
nylon wool columns (Robbins Scientific Corp., Sunnyvale, Calif.).
The enriched T cells were washed in complete medium (RPMI 1640
supplemented with 10% FCS, 15 mM HEPES buffer, 2 mM glutamine, 0.1
mM non-essential amino acids, 50 units/ml penicillin, 50 units/ml
streptomycin, 50 .mu.M 2-mercaptoethanol, and 1 mM sodium pyruvate)
and dispersed into 96-well flat-bottom microtiter plates (Falcon;
Becton Dickinson, Lincoln Park, N.J.) at a concentration of
1.times.10.sup.5/well.
[0056] The T lymphocytes were then incubated in the presence of
naive syngeneic splenocytes (5.times.10.sup.5/well) as antigen
presenting cells (APC). Stimulated wells contained either OVA
protein (100,.mu.g/ml), OVA.sub.257-264 peptide (100 .mu.g/ml), or
concanavalin A (Con A; 2.5 .mu.g/ml). Control wells contained only
T cells and APC in complete medium. All cultures were in a final
volume of 200 pi and were incubated at 37.degree. C. in 5% CO.sub.2
for either 2 days (Con A) or 5 days (antigen stimulants). Cultures
were pulsed with 1 .mu.Ci/well [.sup.3H]thymidine (DuPont New
England Nuclear, Wilmington, Del.) for the final 18 to 24 hours.
Cultures were harvested using a PHD cell harvester (Cambridge
Technology, Cambridge, Mass.) and incorporated radioactivity was
quantitated by liquid scintillation spectroscopy (LS 60001C,
Duarte, Calif.). The results of triplicate wells were averaged and
are reported as a stimulation index (SI) calculated by the
following formula:
SI=stimulated wells (cpm)/control wells (cpm)
[0057] In vitro Stimulation of CTL
[0058] Primary CTL Cultures
[0059] Splenocytes (25.times.10.sup.6) harvested from each
experimental group, pooled from the spleens of three animals per
group, were incubated in 10 ml of complete RPMI (10% FCS, 15 mM
HEPES buffer, 2 mM glutamine, 0.1 mM non-essential amino acids, 50
units/ml penicillin, 50 units/ml streptomycin, 50 .mu.M
2-mercaptoethanol, and 1 mM sodium pyruvate) in upright 25 cm.sup.2
flasks at 37.degree. C. in 5% CO.sub.2 in the presence of 5
.mu.g/ml OVA.sub.257-264 peptide. Long-term CTL Lines
[0060] Primary CTL cultures were harvested after seven days, and
viable lymphocytes were recovered by centrifugation over a Ficoll
gradient (d=1.08 g/ml; Organon Teknika Corp., Durham, N.C.). The
recovered cells were restimulated in 24-well flat-bottom plates
(Corning Costar Corp., Cambridge, Mass.) containing
0.5.times.10.sup.6 lymphocytes, 5.times.10.sup.6 irradiated (2,000
rads) syngeneic C57BL/6 spleen cells, 5 .mu.g/ml OVA.sub.257-264
peptide, and 10 units/ml recombinant human interleukin-2 (IL-2)
(Cetus Corp., Emeryville, Calif.). Subsequent weekly restimulations
of antigen specific CTL were performed in the same manner with the
exception of peptide dose. After 8 weeks of in vitro stimulation,
the peptide concentration was reduced to 2 .mu.g/ml.
[0061] Cytotoxicity Assays
[0062] Four hour .sup.51Cr release assays were performed. Target
cells (tumor cells) were labeled with 50 .mu.Ci
Na.sup.51CrO.sub.4/1.times.10.s- up.6 cells for 90 minutes. Target
cells (1.times.10.sup.4) were labeled in 50 .mu.l of complete RPMI
medium and were added to the wells of a 96-well U-bottom plate
(Corning Costar Corp.). When appropriate, target cells were
incubated for 30 minutes at 37.degree. C. in 5% CO.sub.2 with one
or more of the following before the addition of T cell effectors:
OVA.sub.257-264 peptide, anti-CD8 antibody (supernatant from the
2.43 hybridoma), or anti-CD4 antibody (supernatant from the GK 1.5
hybridoma). Effector cells were added to the targets in 50 .mu.l of
complete medium. The plates were then incubated at 37.degree. C. in
5% CO.sub.2 for four hours. Following incubation, supernatants were
harvested using Skatron harvesting frames (Skatron, Inc., Sterling,
Va.). The release of radioactivity was quantitated using a gamma
counter (Beckman Instruments) and the percent specific lysis was
calculated using the equation: 1 % specific lysis = experimental (
cpm ) - spontaneous release ( cpm ) maximum release ( cpm ) -
spontaneous release ( cpm ) .times. 100
[0063] Results were reported as the mean plus or minus the standard
error of the mean of triplicate cultures.
[0064] Spontaneous release was calculated from wells to which 100
ml of medium had been added in the absence of T cell effectors.
Maximum release was calculated from wells to which a solution of 2%
Triton X-100 was added. Flow Cytometry
[0065] Lymphocytes were harvested and washed three times with cold
Dulbecco's phosphate-buffered saline (DPBS) containing Ca.sup.2+
and Mg.sup.2+ supplemented with 5% fetal bovine serum (FBS). Cells
were incubated on ice for 45 minutes with either fluorescein
isothiocyanate (FITC)-conjugated anti-mouse CD2, CD3, CD4, CD8,
CD28, CD11a/CD18, and .alpha./.beta.T cell receptor (TCR), or the
appropriate isotype control FITC-conjugated rat IgG2aK, rat
IgG2b.lambda., or hamster IgG antibody (PharMingen, San Diego,
Calif.), and then washed twice with DPBS solution free of Ca.sup.2+
and Mg.sup.2+. Data from 10,000 live cells/sample were analyzed
using flow cytometric analysis as known to one skilled in the art
with a Becton Dickinson FACScan.RTM. flow cytometer using an
excitation wavelength of 488 nm and a band pass filter of 530
nm.
[0066] Lymphoproliferative Analysis
[0067] FIG. 2A and FIG. 2B show the results of a
lymphoproliferative analysis. As shown in FIG. 2A, and to determine
if oral immunization with the model protein OVA could result in the
activation of a cellular immune response, C57BL/6 mice were
immunized three times with enterocoated microspheres containing OVA
protein (microsphere-OVA) at concentrations of 12.5 .mu.g/ml, 25
.mu.g/ml, 50 .mu.g/ml and 100 .mu.g/ml (hatched bars). To compare
the immune response generated following oral immunization with OVA
to that of parenteral immunization with the same antigen, OVA
protein was emulsified in DETOX-PC.RTM. adjuvant and administered
subcutaneously to a second group of C57BL/6 mice (solid bars). A
third group of C57BL/6 mice received a placebo microsphere by oral
administration (open bars). Lymphocyte proliferation was assessed
by measuring [.sup.3H]thymidine incorporation.
[0068] As shown in FIG. 2A, T cells from mice receiving 100
.mu.g/ml microsphere-OVA orally had a stimulation index of 38.3,
while T cells from mice immunized with OVA protein in adjuvant had
a stimulation index of 9.1. Naive splenocytes did not proliferate
in the presence of OVA protein. As shown in FIG. 2B, lymphocytes
from each group showed strong stimulation indices upon non-specific
mitogen stimulation with 2.5 .mu.g/ml Con A.
EXAMPLE 2
[0069] A CTL immune response in mice that had been orally immunized
with enterocoated microsphere-OVA generated an antigen-specific T
cell line. Purified splenocytes from mice immunized with OVA,
either orally in microspheres or, as a control, subcutaneously in
an emulsion with DETOX-PC.RTM. adjuvant, were cultured in vitro in
the presence of OVA.sub.257-264 peptide, irradiated syngeneic
splenocytes as APC, and IL-2. The cell lines were maintained on
seven-day cycles of in vitro stimulation (IVS). The ability of the
cell lines to lyse target cells in an antigen-dependent manner was
evaluated five days into the IVS cycle using a four-hour .sup.51Cr
release assay. The EL4 (H-2K.sup.b) cell line was used as a target
cell in these assays. EL4 cells were pre-pulsed with
OVA.sub.257-264 peptide prior to the addition of T cell effector
cells into the assay. All data are at a 20:1 effector:target
ratio.
[0070] As shown in FIG. 3A and FIG. 3B, the emergence of
antigen-specific lysis was evident after only three cycles of IVS.
FIG. 3A shows T cell effectors from mice immunized by subcutaneous
administration of OVA emulsified in DETOX-PC.RTM. adjuvant. FIG. 3B
shows T cell effectors from mice immunized by oral administration
of microsphere-OVA. Closed circles represent EL4 cells pre-pulsed
with 25 .mu.g/ml OVA.sub.257-264 CTL epitope peptide. Open circles
represent non-pulsed EL4 cells.
[0071] While antigen-specific lysis was evident after three cycles
of IVS, non-specific lysis of EL4 cells was also observed at this
time point. Following six cycles of IVS, non-specific lysis of EL4
cells had dropped substantially (about 10% to about 20%). At the
eighth cycle of IVS, both cell lines were approaching higher (about
50% to about 60%) levels of antigen-specific lysis with very low
levels (less than about 10%) of non-specific lysis.
[0072] As shown in FIG. 4, the strength of the CTL lines derived
from immunized animals was evaluated as a function of
effector:target ratio. EL4 cells were pre-pulsed with 25 .mu.g/ml
OVA.sub.257-264 peptide. Closed circles represent microsphere-OVA.
Closed squares represent OVA emulsified in DETOX-PC.RTM. adjuvant.
Crosses represent placebo microspheres and open triangles represent
non-specific .sup.51Cr uptake of non-peptide pulsed EL4 cells.
[0073] Both CTL lines could be titrated through a range of
effector:target ratios. When splenocytes from animals that had been
administered placebo microspheres were cultured under the same
conditions as the experimental cell lines, they could not be in
vitro activated to recognize peptide pulsed target cells. This
observation also demonstrated that the experimental cell lines
acquired their antigen specificity via in vivo activation following
oral or parenteral immunization with OVA, and not as a result of in
vitro culture conditions.
[0074] To confirm that the cell lines derived from each group of
immunized animals lysed tumor cells in a CD8+ T cell dependent
fashion, antibody blocking experiments were performed. FIG. 5A and
FIG. 5B show CD8+ T cell dependence of antigen-specific target cell
lysis. FIG. 5A shows a four hour .sup.51Cr release assay at a 40:1
effector:target ratio using OVA.sub.257-264 pulsed EL4 target
cells, to determine dependence of CD8+ T cells on the observed
target cell lysis by the CTL line derived from animals immunized by
subcutaneous administration of OVA in adjuvant. FIG. 5B shows a
four hour .sup.51Cr release assay at a 20:1 effector: target ratio
using OVA.sub.257264 pulsed EL4 target cells, to determine
dependence of CD8+ T cells on the observed target cell lysis by the
CTL line derived from animals immunized by oral administration of
microsphere-OVA.
[0075] As shown in FIG. 5A and FIG. 5B, in four hour .sup.51Cr
release assays, the supernatant from either the hybridoma GK1.5,
secreting anti-CD4 antibody, or the hybridoma 2.43, secreting
anti-CD8 antibody, was incubated with T cells prior to their
addition to OVA.sub.257-264 pulsed EL4 target cells. In the
presence of anti-CD8 antibody, the antigen-specific tumor cell
lysis was inhibited. Conversely, the presence of anti-CD4 antibody
resulted in minimal (about 1% to about 10%) inhibition of T cell
mediated antigen-specific cell lysis. The lytic activity of both T
cell lines was eliminated when the T cells were pre-incubated with
the supernatant of the 2.43 hybridoma that contains anti-CD8
antibody. Preincubation of the T cells with GK 1.5 hybridoma
supernatant containing anti-CD4 antibody did not cause a major
decrease in the lytic activity of the cell line.
[0076] FACS Analysis
[0077] The presence of T cell surface markers on OVA-derived cell
lines was analyzed by flow cytometry. Table 1 shows phenotypic
characterization of T cell lines following eight cycles of IVS. The
cell lines were derived from splenocytes of mice that had been
immunized with either microsphere-OVA or OVA in adjuvant as
previously described.
1TABLE 1 % Positive Cells Cellular (mean fluorescence intensity)
Determinant Ovalbumin-DETOX-PC .RTM. Microsphere Ovalbumin CD3
95.55 (23.32) 99.18 (77.94) CD4 57.69 (62.37) 8.02 (52.63) CD8
49.68 (138.16) 92.69 (174.30) CD2 78.82 (19.98) 69.14 (26.37) CD28
12.98 (32.01) 60.99 (18.38) CD11a/CD18 99.58 (157.77) 98.22 (89.00)
.alpha./.beta. TCR 64.34 (16.53) 77.33 (21.78)
[0078] As shown in Table 1, both cell lines had a population of
greater than about 95% T cells as identified by the CD3 cell
surface molecule. The T cell line derived from lymphocytes cultured
from mice immunized with OVA in adjuvant contained 49.6% CD8.sup.+
T cells, and the cell line derived from lymphocytes cultured from
mice orally immunized with microsphere-OVA contained 92.7%
CD8.sup.+ T cells. Both cell lines were shown to express the
costimulatory molecule receptors CD2 and CD28, in addition to the
integrin molecule CD11a/CD18. The cultured T cells from both groups
of immunized animals also expressed the usage of an .alpha./.beta.
T cell receptor. These data help to illustrate that the T cells
activated through oral microsphere immunization with the protein
antigen OVA are phenotypically similar to the repertoire activated
following parenteral immunization with the same antigen.
[0079] The microspheres of the present invention modulate an immune
response. The response may encompass a general enhanced production
of T.sub.H1 cells, T.sub.H2 cells and cytotoxic T lymphocyte (CTL)
subsets, or an enhanced shift from a T.sub.H2 type response to a
T.sub.H1 type response, or an enhanced shift from a T.sub.H1 type
response to a T.sub.H2 type response, or an enhanced
differentiation of pre-CTL to CTL. The immunogen may be a peptide,
a protein fragment, a protein, a DNA, and/or an RNA, and may be a
gene, a gene fragment or a vaccine. The therapeutic or prophylactic
agents encompass immunogens, immunotherapy agents or gene therapy
agents, either separately or in combination, that may be orally
delivered in enteric microencapsulated formulations as bound to an
inert particle having a size greater than about 35 mesh and in the
form of a substrate bead, granule, powder, or crystal.
[0080] It will be appreciated that the delivery system composition
and methods disclosed herein can be used prophylactically and
therapeutically in a wide array of conditions. Thus, the
embodiments of the present invention shown and described in the
specification are only preferred embodiments of the inventor who is
skilled in the art and are not limiting in any way. Various
changes, modifications or alterations to these embodiments may be
made or resorted to without departing from the spirit of the
invention and the scope of the following claims.
* * * * *