U.S. patent application number 11/015734 was filed with the patent office on 2005-11-24 for use of vitelline protein b as a microencapsulating additive.
This patent application is currently assigned to The Texas A&M University System. Invention is credited to Carson, Kenneth H., Ficht, Allison, Sheffield, Cynthia, Waite, John Herbert.
Application Number | 20050260258 11/015734 |
Document ID | / |
Family ID | 35375419 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260258 |
Kind Code |
A1 |
Ficht, Allison ; et
al. |
November 24, 2005 |
Use of vitelline protein B as a microencapsulating additive
Abstract
The present invention includes compositions and methods for the
use of an encapsulation additive having between about 0.1 to about
30 percent isolated and purified vitelline protein B to provide for
mixed and extended release formulations.
Inventors: |
Ficht, Allison; (College
Station, TX) ; Carson, Kenneth H.; (Bryan, TX)
; Waite, John Herbert; (Santa Barbara, CA) ;
Sheffield, Cynthia; (College Station, TX) |
Correspondence
Address: |
CHALKER FLORES, LLP
12700 PARK CENTRAL, STE. 455
DALLAS
TX
75251
US
|
Assignee: |
The Texas A&M University
System
College Station
TX
|
Family ID: |
35375419 |
Appl. No.: |
11/015734 |
Filed: |
December 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60530721 |
Dec 18, 2003 |
|
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Current U.S.
Class: |
424/450 ;
424/185.1 |
Current CPC
Class: |
A61K 9/5036 20130101;
A61K 9/1652 20130101; A61K 2039/6031 20130101; A61K 39/0003
20130101; A61K 2039/575 20130101; A61K 38/17 20130101; A61P 31/00
20180101; A61K 9/0019 20130101; A61K 9/1658 20130101; Y02A 50/30
20180101; A61K 9/5052 20130101; A61P 31/04 20180101; A61P 37/04
20180101; A61K 38/21 20130101; A61K 9/5089 20130101; A61P 31/12
20180101; A61K 39/08 20130101; A61K 2039/542 20130101; A61P 31/10
20180101; A61K 38/17 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/450 ;
424/185.1 |
International
Class: |
A61K 039/00; A61K
009/127; A61K 009/50 |
Goverment Interests
[0001] The government may own rights in the present invention
pursuant to grant number NCC-1-02038 from the National Aeronautics
and Space Administration (NASA); contract #0300125 from the US
Geological Survey, Department of the Interior, National Park
Service; and contract# DAMD17-95-C-5048 from the U.S. Army Medical
Research and Materiel Command.
Claims
What is claimed is:
1. An encapsulation additive comprising between about 0.1 to about
90 percent isolated and purified vitelline protein B.
2. The additive of claim 1, wherein the vitelline protein B
comprises a Trematode sp. protein.
3. The additive of claim 1, wherein the additive is non-antigenic,
is resistant to acid pH, resistant to basic pH or combinations
thereof.
4. The additive of claim 1, wherein the additive slows the release
of an active agent to between about 1 hour to about 8 hours,
between about 1 hour to about 2 weeks, and between about 1 hour to
about 6 months.
5. The additive of claim 4, wherein the one or more active agents
comprise a pharmaceutical agent, an enzyme, a cytokine, a growth
promoting agent, an antibody, an antigen, a vaccine, a cell, a
live-attenuated pathogen, a heat-killed pathogen, a virus, a
bacteria, a fungi, a peptide, a carbohydrate, a nucleic acid, a
lipid, mixtures and combinations thereof.
6. The additive of claim 1, wherein the vitelline protein B is
encapsulated by coacervation, oil-and-water emulsion, liposomes,
spray freezing, spray drying, mixed with alginate or combinations
thereof.
7. The additive of claim 1, wherein the formulation is formulated
for administration to a mammal in need of a therapeutic effective
amount of an active agent.
8. The additive of claim 1, wherein the one or more active agents
is selected from the group consisting of protein, peptide,
carbohydrate, polysaccharide, glycoprotein, lipid, hormone, growth
factor, cytokine, interferon, receptor, antigen, allergen,
antibody, antiviral, antifungal, antihelminthic, substrate,
metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient,
toxin, poison, explosive, pesticide, chemical warfare agent,
biowarfare agent, biohazardous agent, infectious agent, prion,
radioisotope, vitamin, heterocyclic aromatic compound, carcinogen,
mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste
product, contaminant, heavy metal, virus, bacterium, Salmonella,
Streptococcus, Brucella, Legionella, E. coli, Giardia,
Cryptosporidium, Rickettsia, spore, mold, yeast, algae, amoebae,
dinoflagellate, unicellular organism, pathogen, cell, combinations
and mixtures thereof, that is encapsulated in a mixed release
polymer comprising between about 0.1 to 26 weight percent isolated
and purified vitelline protein B.
9. An extended release pharmaceutical formulation comprising: one
or more active agents microencapsulated in a mixed release polymer
and between about 0.1 to 26 percent isolated and purified vitelline
protein B.
10. The formulation of claim 9, wherein the polymer is selected
from the group consisting of cellulose, ethylcellulose,
methylcellulose, propylcellulose, methoxypropylcellulose, cellulose
nitrate, poly(vinyl alcohol), poly(vinyl chloride), polystyrene,
polyethylene, polypropylene, poly(ethylene-co-vinyl acetate),
poly(hydroxybutyric acid), poly(hydroxyvalerianic
acid-co-hydroxybutyric acid), poly(lactic acid), poly(glycolic
acid), poly(lactic acid-co-glycolic acid),
poly(epsilon(-caprolactones),
poly(epsilon(-caprolactone-co-DL-lactic acid), poly(maleic
anhydride), polyamides, gelatin, chitosan, collagen,
poly(hydroxyalkyl)-L-glutamines,
poly(gamma(-ethyl-L-glutaminate-co-gluta- mic acid),
poly(L-leucine-co-L-aspartic acid), poly(proline-co-glutamic acid),
poly(alkyl 2-cyanoacrylates), polyurethanes, poly(methyl
methacrylate), poly(methyl methacrylate-co-methacrylic acid) and
poly(methacrylate-co-hydroxypropyl methacrylate).
11. The formulation of claim 9, wherein the vitelline protein B
comprises a Trematode sp. protein.
12. The formulation of claim 9, wherein the additive is
non-antigenic, is resistant to acid and base pH or both.
13. The formulation of claim 9, wherein the additive slows the
release of an active agent to between about 1 hour to about 8
hours, between about 1 hour to about 2 weeks, and between about 1
hour to about 6 months.
14. The formulation of claim 9, wherein the one or more active
agents comprise a pharmaceutical agent, an enzyme, a cytokine, a
growth promoting agent, an antibody, an antigen, a vaccine, a cell,
a live-attenuated pathogen, a heat-killed pathogen, a virus, a
bacteria, a fungi, a peptide, a carbohydrate, a nucleic acid, a
lipid, mixtures and combinations thereof.
15. The formulation of claim 9, wherein the vitelline protein B is
combined by coacervation.
16. The formulation of claim 9, wherein the vitelline protein B is
added into an oil-and-water emulsion.
17. A method of encapsulation comprising mixing one or more active
agents with one or more cross-linkable monomers and a vitelline
protein B, wherein the vitelline protein B modifies the release
profile of the one or more active agents.
18. A method of making an extended release formulation comprising
the steps of: mixing one or more active agents with alginate and
vitelline protein B, wherein the vitelline protein B modifies the
release profile of one or more of the active agents.
19. The method of claim 18, wherein the one or more active agents
are released for between about 1 hour to about 8 hours, between
about 1 hour to about 2 weeks, and between about 1 hour to about 6
months.
20. The method of claim 18, wherein the vitelline protein B
comprises a Trematode sp. protein.
21. The method of claim 18, wherein the vitelline protein B
sequence has been scrambled but maintains about the same amino acid
ratio.
22. The method of claim 18, wherein the one or more active agents
comprise a pharmaceutical agent, an enzyme, a cytokine, a growth
promoting agent, an antibody, an antigen, a vaccine, a cell, a
live-attenuated pathogen, a heat-killed pathogen, a virus, a
bacteria, a fungi, a peptide, a carbohydrate, a nucleic acid, a
lipid, mixtures and combinations thereof.
23. The method of claim 18, wherein the vitelline protein B is
combined by coacervation, an oil-and-water emulsion, liposomes,
spray freezing, spray drying, mixed with alginate or combinations
thereof.
24. The method of claim 18, wherein the formulation is administered
to a mammal in need of a therapeutic effective amount of the one or
more active agents.
25. The method of claim 18, further comprising mixing poly-L lysine
with the vitelline protein B, wherein the ratio of the poly-L
lysine to vitelline protein B is between about 30:70 to 70:30
weight to weight.
26. A mixed release pharmaceutical formulation comprising: one or
more pharmaceutical agents encapsulated with a mixed release
polymer comprising between about 1 to 26 weight percent isolated
and purified vitelline protein B.
27. The mixed release formulation of claim 26, wherein the one or
more pharmaceutical agents is selected from wherein the one or more
agents may be steroids, respiratory agents, sympathomimetics, local
anesthetics, antimicrobial agents, antiviral agents, antifungal
agents, antihelminthic agents, insecticides, antihypertensive
agents, antihypertensive diuretics, cardiotonics, coronary
vasodilators, vasoconstrictors, .beta.-blockers, antiarrhythmic
agents, calcium antagonists, anti-convulsants, agents for
dizziness, tranquilizers, antipsychotics, muscle relaxants, drugs
for Parkingson's disease, respiratory agents, hormones,
non-steroidal hormones, antihormones, vitamins, antitumor agents,
miotics, herb medicines, antimuscarinic, interfereons, immunokines,
cytokines, muscarinic cholinergic blocking agents, mydriatics,
psychic energizers, humoral agents, antispasmodics, antidepressant
drugs, anti-diabetics, anorectic drugs, anti-allergenics,
decongestants, expectorants, antipyretics, antimigrane,
anti-malarials, anti-ulcerative, peptides, anti-estrogen,
anti-hormone agents, antiulcer agents, anesthetic agent, drugs
having an action on the central nervous system, protein, peptide,
carbohydrate, polysaccharide, glycoprotein, lipid, hormone, growth
factor, cytokine, interferon, receptor, antigen, allergen,
antibody, substrate, metabolite, cofactor, inhibitor, drug,
pharmaceutical, nutrient, toxin, poison, explosive, pesticide,
chemical warfare agent, biowarfare agent, biohazardous agent,
infectious agent, prion, radioisotope, vitamin, heterocyclic
aromatic compound, carcinogen, mutagen, narcotic, amphetamine,
barbiturate, hallucinogen, waste product, contaminant, heavy metal,
virus, bacterium, Salmonella, Streptococcus, Brucella, Legionella,
E. coli, Giardia, Cryptosporidium, Rickettsia, spore, mold, yeast,
algae, amoebae, dinoflagellate, unicellular organism, pathogen,
cell, combinations and mixtures thereof, that is encapsulated in a
mixed release polymer comprising between about 1 to 26 weight
percent isolated and purified vitelline protein B.
28. The formulation of claim 26, wherein the polymer is selected
from the group consisting of poly(ethylene glycol), poly(ethylene
oxide), poly(vinyl alcohol), poly(vinylpyrrolidone),
poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)
block copolymers, polysaccharides, carbohydrates, proteins, and
combinations thereof.
29. A vaccine comprising: one or more antigens encapsulated in a
mixed release comprising between about 1 to 26 weight percent
isolated and purified vitelline protein B, the antigen selected
from protein, peptide, carbohydrate, polysaccharide, glycoprotein,
lipid, hormone, growth factor, cytokine, interferon, receptor,
antigen, allergen, antibody, substrate, metabolite, cofactor,
inhibitor, drug, pharmaceutical, nutrient, toxin, poison,
explosive, pesticide, chemical warfare agent, biowarfare agent,
biohazardous agent, infectious agent, prion, radioisotope, vitamin,
heterocyclic aromatic compound, carcinogen, mutagen, narcotic,
amphetamine, barbiturate, hallucinogen, waste product, contaminant,
heavy metal, virus, bacterium, Salmonella, Streptococcus, Brucella,
Legionella, E. coli, Giardia, Cryptosporidium, Rickettsia, spore,
mold, yeast, algae, amoebae, dinoflagellate, unicellular organism,
pathogen, cell, combinations and mixtures thereof.
30. An adhesive comprising between about 0.1 to about 90 percent
isolated and purified vitelline protein B.
31. A method of treating an animal, the method comprising
administering the extended release formulation of claim 9 to a
mammal.
32. A bent core mesogen piezoelectric component comprising a VpB
protein.
Description
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for
extended release encapsulation using additives, and more
particularly, to vitelline proteins as microencapsulation additives
and uses thereof.
BACKGROUND OF THE INVENTION
[0003] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/530,721, filed Dec. 18, 2003. Without
limiting the scope of the invention, its background is described in
connection with polymeric encapsulation of compounds.
[0004] The encapsulation of pharmaceuticals and other compounds is
of continuing interest, both in industry and in academia.
Encapsulation may be used to modify the time-release properties of
a material, to alter the solubility of a material and/or to provide
other desirable property modifications. Polymers, biopolymers and
peptides have been explored for their suitability as
microencapsulation agents.
[0005] Microcapsules include, generally, a core material (liquid or
solid) encased in a specialized coating. Microencapsulation
technology has found widespread use in the pharmaceutical area, for
example, coating of drugs to extend or delay their release or
target their release to a specific area of the digestive tract.
Other interesting applications include encapsulation of ink in
carbonless carbon paper, encapsulation of perfumes in `scratch and
sniff` types of promotionals and encapsulation of living pancreatic
cells to treat diabetes. In the case of living cells, the pore size
of the microcapsule is selected to prevent the passage of host
antibodies while allowing the free exchange of insulin and
glucagon. Microcapsules have also found application in the food,
cleaning, adhesives, fertilizers and aerospace industries to name a
few
[0006] Albumin has been used in the preparation of microcapsules.
Two technologies have been used--coacervation, or oil in water
emulsions. These techniques have been the source of many
publications, including Zimmer et al. (J. Controlled Release 33:
3146 (1995)); Cremers et al. (Biomaterials 15(1): 38-48 (1994));
Deasy (Microencapsulation and Related Drug Processes; Marcel
Dekker, Inc., NY (1984)); and Tomlinson, E. and Burger, J. J.
Methods Enzymol. 112: 2743 (1985)). Despite the efforts made
towards developing microencapsulation methods, there still exists a
need for novel and effective materials for the encapsulation of
various materials.
SUMMARY OF THE INVENTION
[0007] The compositions and methods of the present invention
include, generally, an encapsulation additive comprising between
about 0.1 to about 30 percent isolated and purified vitelline
protein B. The present inventors recognized that a key aspect of
trematode infection and survival is the avoidance of host immune
responses. Studies as to the causes for evasion of host immune
surveillance led to the isolation and characterization of proteins
from trematode eggshells. Upon isolation of the genes for the
trematode proteins, vpA, vpB and vpC and isoforms of the same, it
was discovered, as disclosed herein that the vpB protein, in
particular, is stable at a wide range of pHs, is protease resistant
and non-immunogenic. As such, the present investigators used the
vpB protein as an additive for the encapsulation and extended
release of, small molecules, antigens and the like.
[0008] More particularly, the encapsulation additive of the present
invention may include the vitelline protein B from a Trematode sp.
The additive is generally non-antigenic, is resistant to acid and
base pH or both and may allow for the slow release of an active
agent to between about 1 hour to about to about 8 hours, between
about 1 hour to about 2 weeks, and between about 1 hour to about 6
months. Examples of the one or more active agents may include a
pharmaceutical agent, an enzyme, a cytokine, a growth promoting
agent, an antibody, an antigen, a vaccine, a cell, a
live-attenuated pathogen, a heat-killed pathogen, a virus, a
bacteria, a fungi, a peptide, a carbohydrate, a nucleic acid, a
lipid, mixtures and combinations thereof. The vitelline protein B
may be formed into a capsule when combined by coacervation, added
into an oil-and-water emulsion, liposomes, spray freezing, spray
drying, as a component of an alginate microcapsule or combinations
thereof. Other examples of active agents include: protein, peptide,
carbohydrate, polysaccharide, glycoprotein, lipid, hormone, growth
factor, cytokine, interferon, receptor, antigen, allergen,
antibody, substrate, metabolite, cofactor, inhibitor, drug,
pharmaceutical, nutrient, toxin, poison, explosive, pesticide,
chemical warfare agent, biowarfare agent, biohazardous agent,
infectious agent, prion, radioisotope, vitamin, heterocyclic
aromatic compound, carcinogen, mutagen, narcotic, amphetamine,
barbiturate, hallucinogen, waste product, contaminant, heavy metal,
virus, bacterium, Salmonella, Streptococcus, Brucella, Legionella,
E. coli, Giardia, Cryptosporidium, Rickettsia, spore, mold, yeast,
algae, amoebae, dinoflagellate, unicellular organism, pathogen,
cell, combinations and mixtures thereof, that is encapsulated in a
mixed release polymer comprising between about 0.1 to about 26,
about 0.1 to about 30, or even about 0.1 to about 90 weight percent
isolated and purified recombinant vitelline protein B.
[0009] Yet another embodiment of the present invention may be a
mixed release pharmaceutical formulation with one or more active
agents microencapsulated in a mixed release polymer and between
about 0.1 to 26 percent isolated and purified vitelline protein B.
A polymer selected from the group consisting of cellulose,
ethylcellulose, methylcellulose, propylcellulose,
methoxypropylcellulose, cellulose nitrate, poly(vinyl alcohol),
poly(vinyl chloride), polystyrene, polyethylene, polypropylene,
poly(ethylene-co-vinyl acetate), poly(hydroxybutyric acid),
poly(hydroxyvalerianic acid-co-hydroxybutyric acid), poly(lactic
acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid),
poly(epsilon(-caprolactones),
poly(epsilon(-caprolactone-co-DL-lactic acid), poly(maleic
anhydride), polyamides, gelatin, chitosan, collagen,
poly(hydroxyalkyl)-L-glutamines,
poly(gamma(-ethyl-L-glutaminate-co-gluta- mic acid),
poly(L-leucine-co-L-aspartic acid), poly(proline-co-glutamic acid),
poly(alkyl 2-cyanoacrylates), polyurethanes, poly(methyl
methacrylate), poly(methyl methacrylate-co-methacrylic acid) and
poly(methacrylate-co-hydroxypropyl methacrylate).
[0010] Other examples of active agents may include a pharmaceutical
agent, an enzyme, a cytokine, a growth promoting agent, an
antibody, an antigen, a vaccine, a cell, a live-attenuated
pathogen, a heat-killed pathogen, a virus, a bacteria, a fungi, a
peptide, a carbohydrate, a nucleic acid, a lipid, mixtures and
combinations thereof.
[0011] The present invention may also include a method of
encapsulation that includes the steps of mixing one or more active
agents with one or more cross-linkable monomers and a vitelline
protein B, wherein the vitelline protein B modifies the release
profile of the one or more active agents. Alternatively, the method
of making an extended release formulation may include the steps of
mixing one or more active agents with alginate and vitelline
protein B, wherein the vitelline protein B modifies the release
profile of one or more of the active agents. The one or more active
agents are released for between about I hour to about 8 hours,
between about I hour to about 2 weeks, and between about 1 hour to
about 6 month and the vitelline protein B comprises a Trematode sp.
protein that is non-antigenic, is resistant to acid pH, resistant
to basic pH or combinations thereof. The method may also include
the step of mixing poly-L lysine with the vitelline protein B,
wherein the ratio of the poly-L lysine to vitelline protein B is
between about 30:70 to 70:30 weight to weight.
[0012] Yet another embodiment of the present invention is a mixed
release pharmaceutical formulation in which one or more one or more
pharmaceutical agents is encapsulated in a mixed release polymer
comprising between about 0.1 to 26 weight percent isolated and
purified vitelline protein B. Examples of pharmaceutical agents
include: steroids, respiratory agents, sympathomimetics, local
anesthetics, antimicrobial agents, antiviral agents, antifungal
agents, antihelminthic agents, insecticides, antihypertensive
agents, antihypertensive diuretics, cardiotonics, coronary
vasodilators, vasoconstrictors, P-blockers, antiarrhythmic agents,
calcium antagonists, anti-convulsants, agents for dizziness,
tranquilizers, antipsychotics, muscle relaxants, drugs for
Parkingson's disease, respiratory agents, hormones, non-steroidal
hormones, antihormones, vitamins, antitumor agents, miotics, herb
medicines, antimuscarinic, interfereons, immunokines, cytokines,
muscarinic cholinergic blocking agents, mydriatics, psychic
energizers, humoral agents, antispasmodics, antidepressant drugs,
anti-diabetics, anorectic drugs, anti-allergenics, decongestants,
expectorants, antipyretics, antimigrane, anti-malarials,
anti-ulcerative, peptides, anti-estrogen, anti-hormone agents,
antiulcer agents, anesthetic agent, drugs having an action on the
central nervous system or combinations thereof. The polymer may be
a biocompatible and/or biodegradable polymer selected from:
poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, polysaccharides,
carbohydrates, proteins and combinations thereof.
[0013] A mixed release pharmaceutical formulation using the vpB
additive of the present invention may include one or more active
agents selected from protein, peptide, carbohydrate,
polysaccharide, glycoprotein, lipid, hormone, growth factor,
cytokine, interferon, receptor, antigen, allergen, antibody,
substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical,
nutrient, toxin, poison, explosive, pesticide, chemical warfare
agent, biowarfare agent, biohazardous agent, infectious agent,
prion, radioisotope, vitamin, heterocyclic aromatic compound,
carcinogen, mutagen, narcotic, amphetamine, barbiturate,
hallucinogen, waste product, contaminant, heavy metal, virus,
bacterium, Salmonella, Streptococcus, Brucella, Legionella, E.
coli, Giardia, Cryptosporidium, Rickettsia, spore, mold, yeast,
algae, amoebae, dinoflagellate, unicellular organism, pathogen,
cell, combinations and mixtures thereof, that is encapsulated in a
mixed release polymer and between about 0.1 to 26 weight percent
isolated and purified vitelline protein B.
[0014] Yet another embodiment of the present invention is a vaccine
in which one or more antigens are encapsulated in a mixed release
with between about 0.1 to 26 weight percent isolated and purified
vitelline protein B. The antigen may be selected from: protein,
peptide, carbohydrate, polysaccharide, glycoprotein, lipid,
hormone, growth factor, cytokine, interferon, receptor, antigen,
allergen, antibody, substrate, metabolite, cofactor, inhibitor,
drug, pharmaceutical, nutrient, toxin, poison, explosive,
pesticide, chemical warfare agent, biowarfare agent, biohazardous
agent, infectious agent, prion, radioisotope, vitamin, heterocyclic
aromatic compound, carcinogen, mutagen, narcotic, amphetamine,
barbiturate, hallucinogen, waste product, contaminant, heavy metal,
virus, bacterium, Salmonella, Streptococcus, Brucella, Legionella,
E. coli, Giardia, Cryptosporidium, Rickettsia, spore, mold, yeast,
algae, amoebae, dinoflagellate, unicellular organism, pathogen,
cell, combinations and mixtures thereof. Yet another embodiment of
the present invention is an adhesive having between about 1 to
about 90 percent isolated and purified vitelline protein B.
[0015] The vpB additive and methods disclosed herein may be used
for the micro and nano-encapsulation of a number of active agents,
e.g., peptides, proteins, aptamers, oligonucleotides,
carbohydrates, lipids, glycolipids, glycoproteins, anti-obesity
drugs, nutraceuticals, corticosteroids, elastase inhibitors,
analgesics, anti-fungals, antibiotics, antibodies, antigens,
oncology therapies, anti-emetics, analgesics, cardiovascular
agents, anti-inflammatory agents, antigens, antihelmintics,
antiarrhythmic agents, antibiotics, antibodies, anticoagulants,
antidepressants, antidiabetic agents, antiepileptics,
antihistamines, antihypertensive agents, antimuscarinic agents,
antimycobacterial agents, antineoplastic agents,
immunosuppressants, antithyroid agents, antiviral agents,
anxiolytics, sedatives, astringents, beta-adrenoceptor blocking
agents, blood products and substitutes, cardiac inotropic agents,
contrast media, corticosteroids, cough suppressants, diagnostic
agents, diagnostic imaging agents, diuretics, dopaminergics,
haemostatics, immunological agents (cytokines, lymphokines), lipid
regulating agents, muscle relaxants, parasympathomimetics,
parathyroid calcitonin and biphosphonates, prostaglandins,
radio-pharmaceuticals, sex hormones, insecticides, anti-allergic
agents, stimulants and anoretics, sympathomimetics, thyroid agents,
vasodilators, and xanthines, and derivatives, salts and
combinations thereof. The skilled artisan will recognize that a
number of agents may be used and/or delivered in combination with
the present invention, including, organic or inorganic molecules
(small or large), second messengers, nucleic acids (natural,
non-natural and derivatives thereof), amino acids (natural,
non-natural and derivatives thereof), carbohydrates (monomeric or
oligomeric), lipids, cells, cell fragments, glycoproteins,
nutrients, vitamins, etc.
[0016] The vpB and related proteins of the present invention may
also be used in a method of treating an animal, the method in which
the mixed release formulation of the present invention is
administered to a mammal. In an alternative embodiment, the vpB
protein may also be used as a bent core mesogen piezoelectric
component.
BRIEF DESCRIPTION OF THE FIGURES
[0017] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0018] FIG. 1 is a map that summarizes the divergence of amino acid
sequence between vpB family members: hatched lines indicate areas
of highly divergent amino acid sequence;
[0019] FIG. 2 is a graph that shows the release of a small
molecule, [3H] glycine, encapsulated using the additive of the
present invention;
[0020] FIG. 3 is a graph that shows the antibody titer of mice
treated via subcutaneous injection of encapsulated tetanus
toxoid;
[0021] FIG. 4 is a graph that shows the antibody titer of mice
treated via subcutaneous injection with botulinum toxin
encapsulated using the present invention and delivered as a
depot;
[0022] FIG. 5 is a graph that shows the immune response to doses of
botulinum neurotoxin A, fragment C trapped in the composite capsule
was dose dependent;
[0023] FIG. 6 is a graph that shows the antibody response of six
mice injected subcutaneously with encapsulated TT (tetanus toxoid)
composite capsules;
[0024] FIG. 7 is a graph that shows cellular immune response as
measured by tritium uptake for the same groups as FIG. 6 at 21
weeks;
[0025] FIG. 8 is a graph that shows the results obtained from Red
deer vaccination studies;
[0026] FIG. 9 is a graph that shows the kinetics of humoral
immunity with subcutaneous vaccination of the present
invention;
[0027] FIG. 10 is a graph of serum antibody levels in Brucella
melitensis immunized mice; and
[0028] FIG. 11 shows the fluorescence profile of green fluorescent
protein released over a one week period as determined through SDS
polyacrylamide gel electrophoresis and fluorescence analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0029] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0030] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0031] The term "additive" as used herein is used to describe the
use of a vpB protein, variants thereof, and proteins and peptides
that have a similar protein composition to produce encapsulants
that affect the release of an agent trapped in or about the
additive, e.g., when the additive is mixed with a polymeric or
other composition to form a micro or nanocapsule. It has been
found, as described in detail hereinbelow, that the Vitelline
protein B is a member of a family of proteins with variable
sequences, however, the amino acid composition is fixed. The
Vitelline protein B may be closed and/or isolated by purification
from, e.g., Fasciola hepatica. Alternatively, the Vitelline protein
B may be made synthetically, by recombinant methods and
combinations thereof.
[0032] The term "immediate release" as used herein is used to
describe a release profile to effect delivery of an active as soon
as possible, that is, as soon as practically made available to an
animal, whether in active form, as a precursor and/or as a
metabolite. Immediate release may also be defined functionally as
the release of over 80 to 90 percent (%) of the active ingredient
within about 60, 90, 100 or 120 minutes or less.
[0033] The terms "extended release" and "delayed release" as used
herein is used to define a release profile to effect delivery of an
active over an extended period of time, defined herein as being
between about 60 minutes and about 2, 4, 6 or even 8 hours.
Extended release may also be defined functionally as the release of
over 80 to 90 percent (%) of the active ingredient after about 60
minutes and about 2, 4, 6 or even 8 hours. Extended release as used
herein may also be defined as making the active ingredient
available to the patient or subject regardless of uptake, as some
actives may never be absorbed by the animal. Various extended
release dosage forms may be designed readily by one of skill in art
as disclosed herein to achieve delivery to both the small and large
intestines, to only the small intestine, or to only the large
intestine, depending upon the choice of coating materials and/or
coating thickness.
[0034] "Extended release" and "delayed release" formulations may be
prepared and delivered using the Vitelline protein B or variants
thereof to control the release of an agent. The Vitelline protein B
protein may act alone or in combination with other compounds that
delay release of an active, e.g., a coating, a capsule or mixture
with controlled, delayed or extended release polymers. Any coatings
should be applied to a sufficient thickness such that the entire
coating does not dissolve in the gastrointestinal fluids at pH
below about 5, but does dissolve at pH about 5 and above. It is
expected that any anionic polymer exhibiting a pH-dependent
solubility profile can be used as an enteric coating in the
practice of the present invention to achieve delivery to the lower
gastrointestinal tract. Polymers and compatible mixtures thereof
may be used to provide the coating for the delayed or the extended
release of active ingredients, and some of their properties,
include, but are not limited to: shellac, also called purified lac,
a refined product obtained from the resinous secretion of an
insect. This coating dissolves in media of pH>7.
[0035] As used herein, the term "enveloped pharmaceutical" means a
capsule, a suppository, a gel cap, a softgel, a lozenge, a sachet
or even a fast dissolving wafer. As used herein the term "carrier"
is used to describe a substance, whether biodegradable or not, that
is physiologically acceptable for human or animal use and may be
pharmacologically active or inactive.
[0036] The vpB additive of the present invention may be used in
conjunction with a wide variety of dosage forms, e.g., solution,
suspension, cream, ointment, lotion, capsule, caplet, softgel,
gelcap, suppository, enema, elixir, syrup, emulsion, film, granule,
gum, insert, jelly, foam, paste, pastille, pellet, spray, troche,
lozenge, disk, magma, poultice, or wafer and the like.
[0037] The vpB additive of the present invention may be used to
delivery active pharmaceutical agents. As used herein,
"pharmaceutically" and/or "pharmacologically acceptable" refer to
molecular entities and/or compositions that do not produce an
adverse, allergic and/or other untoward reaction when administered
to an animal, as appropriate. One distinct advantage of vpB is that
it does not generally cause an adverse, allergic and/or other
untoward reaction when administered to an animal.
[0038] As used herein, "pharmaceutically acceptable carrier" may
include any and/or all solvents, dispersion media, coatings,
antibacterial and/or antifungal agents, isotonic and/or absorption
delaying agents and/or the like. The use of such media and/or
agents for pharmaceutical active substances is well known in the
art. Except insofar as any conventional media and/or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions. For administration,
preparations should meet sterility, pyrogenicity, general safety
and/or purity standards as required by FDA Office of Biologics
standards.
[0039] As used herein, the term "therapeutically effective dosage"
is used to describe the amount that reduces the amount of symptoms
of the condition in the infected subject by at least about 20%,
more preferably by at least about 40%, even more preferably by at
least about 60%, and still more preferably by at least about 80%
relative to untreated subjects. Often, for pediatric doses the
amount will be half or less of the adult dose. For example, the
efficacy of a compound may be evaluated in an animal model system
that may be predictive of efficacy in treating the disease in
humans. Bioactive compounds are administered at a therapeutically
effective dosage sufficient to treat a condition associated with a
condition in a subject.
[0040] The terms "amount," "pharmaceutically effective amount" and
"therapeutically effective amount" as used herein refer to a
quantity or to a concentration as appropriate to the context. The
amount of an active agent or drug that constitutes a
pharmaceutically or therapeutically effective amount varies
according to factors such as the potency of the particular drug,
the route of administration of the formulation, and the mechanical
system used to administer the formulation as will be known to the
skilled artisan. For example, a pharmaceutically or therapeutically
effective amount is that dosage of active agents that when released
is sufficient to effect treatment, when administered to a mammal in
need of such treatment. The therapeutically effective amount will
vary depending upon the subject and disease condition being
treated, the weight and age of the subject, the severity of the
disease condition, the manner of administration and the like, which
can readily be determined by one of ordinary skill in the art. The
amount of active agent and the need and amount of carriers and/or
excipients are disclosed, simply by way of example, by Remington's
Pharmaceutical Sciences, 19th edition, 1995, Ed. Gennaro, relevant
portions incorporated herein by reference. The term "treatment" or
"treating" means any treatment of a disease in a mammal, including:
(i) preventing the disease, that is, causing the clinical symptoms
of the disease not to develop; (ii) inhibiting the disease, that
is, arresting the development of clinical symptoms; and/or (iii)
relieving the disease, that is, causing the regression of clinical
symptoms.
[0041] The additive of the present invention may be used in
conjunction with one or more carriers. As used herein the terms
"carrier" and "pharmaceutically acceptable carrier" include any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0042] As used herein, the term "active ingredient(s),"
"pharmaceutical ingredient(s)," "active agents" and "bioactive
agent" are defined as drugs and/or pharmaceutically active
ingredients. The present invention may be used to encapsulate,
attach, bind or otherwise be used to affect the storage, stability,
longevity and/or release of any of the following drugs as the
pharmaceutically active agent in a composition.
[0043] One or more of the following bioactive agents may be
combined with the vpB additive disclosed herein: Analgesic
anti-inflammatory agents such as, acetaminophen, aspirin, salicylic
acid, methyl salicylate, choline salicylate, glycol salicylate,
1-menthol, camphor, mefenamic acid, fluphenamic acid, indomethacin,
diclofenac, alclofenac, ibuprofen, ketoprofen, naproxene,
pranoprofen, fenoprofen, sulindac, fenbufen, clidanac,
flurbiprofen, indoprofen, protizidic acid, fentiazac, tolmetin,
tiaprofenic acid, bendazac, bufexamac, piroxicam, phenylbutazone,
oxyphenbutazone, clofezone, pentazocine, mepirizole and the
like.
[0044] Drugs having an action on the central nervous system, for
example sedatives, hypnotics, antianxiety agents, analgesics and
anesthetics, such as, chloral, buprenorphine, naloxone,
haloperidol, fluphenazine, pentobarbital, phenobarbital,
secobarbital, amobarbital, cydobarbital, codeine, lidocaine,
tetracaine, dyclonine, dibucaine, cocaine, procaine, mepivacaine,
bupivacaine, etidocaine, prilocaine, benzocaine, fentanyl, nicotine
and the like. Local anesthetics such as, benzocaine, procaine,
dibucaine, lidocaine and the like.
[0045] Antihistaminics or antiallergic agents such as,
diphenhydramine, dimenhydrinate, perphenazine, triprolidine,
pyrilamine, chlorcyclizine, promethazine, carbinoxamine,
tripelennamine, brompheniramine, hydroxyzine, cyclizine, meclizine,
clorprenaline, terfenadine, chlorpheniramine and the like.
Anti-allergenics such as, antazoline, methapyrilene,
chlorpheniramine, pyrilamine, pheniramine and the like.
Decongestants such as, phenylephrine, ephedrine, naphazoline,
tetrahydrozoline and the like.
[0046] Antipyretics such as, aspirin, salicylamide, non-steroidal
anti-inflammatory agents and the like. Antimigrane agents such as,
dihydroergotamine, pizotyline and the like. Acetonide
anti-inflammatory agents, such as hydrocortisone, cortisone,
dexamethasone, fluocinolone, triamcinolone, medrysone,
prednisolone, flurandrenolide, prednisone, halcinonide,
methylprednisolone, fludrocortisone, corticosterone, paramethasone,
betamethasone, ibuprophen, naproxen, fenoprofen, fenbufen,
flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin,
piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate,
phenylbutazone, sulindac, mefenamic acid, meclofenamate sodium,
tolmetin and the like. Muscle relaxants such as, tolperisone,
baclofen, dantrolene sodium, cyclobenzaprine.
[0047] Steroids such as, androgenic steriods, such as,
testosterone, methyltestosterone, fluoxymesterone, estrogens such
as, conjugated estrogens, esterified estrogens, estropipate,
17-.beta. estradiol, 17-.beta. estradiol valerate, equilin,
mestranol, estrone, estriol, 17.beta. ethinyl estradiol,
diethylstilbestrol, progestational agents, such as, progesterone,
19-norprogesterone, norethindrone, norethindrone acetate,
melengestrol, chlormadinone, ethisterone, medroxyprogesterone
acetate, hydroxyprogesterone caproate, ethynodiol diacetate,
norethynodrel, 17-.alpha. hydroxyprogesterone, dydrogesterone,
dimethisterone, ethinylestrenol, norgestrel, demegestone,
promegestone, megestrol acetate and the like.
[0048] Respiratory agents such as, theophilline and
.beta.2-adrenergic agonists, such as, albuterol, terbutaline,
metaproterenol, ritodrine, carbuterol, fenoterol, quinterenol,
rimiterol, solmefamol, soterenol, tetroquinol and the like.
Sympathomimetics such as, dopamine, norepinephrine,
phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine,
propylhexedrine, arecoline and the like.
[0049] Antimicrobial agents including antibacterial agents,
antifungal agents, antimycotic agents and antiviral agents;
tetracyclines such as, oxytetracycline, penicillins, such as,
ampicillin, cephalosporins such as, cefalotin, aminoglycosides,
such as, kanamycin, macrolides such as, erythromycin,
chloramphenicol, iodides, nitrofrantoin, nystatin, amphotericin,
fradiomycin, sulfonamides, purrolnitrin, clotrimazole, miconazole
chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine,
sulfamerazine, sulfamethizole and sulfisoxazole; antivirals,
including idoxuridine; clarithromycin; and other anti-infectives
including nitrofurazone and the like.
[0050] Antihypertensive agents such as, clonidine,
.alpha.-methyldopa, reserpine, syrosingopine, rescinnamine,
cinnarizine, hydrazine, prazosin and the like. Antihypertensive
diuretics such as, chlorothiazide, hydrochlorothrazide,
bendoflumethazide, trichlormethiazide, furosemide, tripamide,
methyldlothiazide, penfluzide, hydrothiazide, spironolactone,
metolazone and the like. Cardiotonics such as, digitalis,
ubidecarenone, dopamine and the like. Coronary vasodilators such
as, organic nitrates such as, nitroglycerine, isosorbitol
dinitrate, erythritol tetranitrate, and pentaerythritol
tetranitrate, dipyridamole, dilazep, trapidil, trimetazidine and
the like. Vasoconstrictors such as, dihydroergotamine,
dihydroergotoxine and the like. .beta.-blockers or antiarrhythmic
agents such as, timolol pindolol, propranolol and the like. Humoral
agents such as, the prostaglandins, natural and synthetic, for
example PGE1, PGE2.alpha., and PGF2.alpha., and the PGE1 analog
misoprostol. Antispasmodics such as, atropine, methantheline,
papaverine, cinnamedrine, methscopolamine and the like.
[0051] Calcium antagonists and other circulatory organ agents, such
as, aptopril, diltiazem, nifedipine, nicardipine, verapamil,
bencyclane, ifenprodil tartarate, molsidomine, clonidine, prazosin
and the like. Anti-convulsants such as, nitrazepam, meprobamate,
phenytoin and the like. Agents for dizziness such as, isoprenaline,
betahistine, scopolamine and the like. Tranquilizers such as,
reserprine, chlorpromazine, and antianxiety benzodiazepines such
as, alprazolam, chlordiazepoxide, clorazeptate, halazepam,
oxazepam, prazepam, clonazepam, flurazepam, triazolam, lorazepam,
diazepam and the like.
[0052] Antipsychotics such as, phenothiazines including
thiopropazate, chlorpromazine, triflupromazine, mesoridazine,
piperracetazine, thioridazine, acetophenazine, fluphenazine,
perphenazine, trifluoperazine, and other major tranqulizers such
as, chlorprathixene, thiothixene, haloperidol, bromperidol,
loxapine, and molindone, as well as, those agents used at lower
doses in the treatment of nausea, vomiting and the like.
[0053] Drugs for Parkingson's disease, spasticity, and acute muscle
spasms such as levodopa, carbidopa, amantadine, apomorphine,
bromocriptine, selegiline (deprenyl), trihexyphenidyl
hydrochloride, benztropine mesylate, procyclidine hydrochloride,
baclofen, diazepam, dantrolene and the like. Respiratory agents
such as, codeine, ephedrine, isoproterenol, dextromethorphan,
orciprenaline, ipratropium bromide, cromglycic acid and the like.
Non-steroidal hormones or antihormones such as, corticotropin,
oxytocin, vasopressin, salivary hormone, thyroid hormone, adrenal
hormone, kallikrein, insulin, oxendolone and the like.
[0054] Vitamins such as, vitamins A, B, C, D, E and K and
derivatives thereof, calciferols, mecobalamin and the like for
dermatologically use. Enzymes such as, lysozyme, urokinaze and the
like. Herb medicines or crude extracts such as, Aloe vera and the
like.
[0055] Antitumor agents such as, 5-fluorouracil and derivatives
thereof, krestin, picibanil, ancitabine, cytarabine and the like.
Anti-estrogen or anti-hormone agents such as, tamoxifen or human
chorionic gonadotropin and the like. Miotics such as pilocarpine
and the like.
[0056] Cholinergic agonists such as, choline, acetylcholine,
methacholine, carbachol, bethanechol, pilocarpine, muscarine,
arecoline and the like. Antimuscarinic or muscarinic cholinergic
blocking agents such as, atropine, scopolamine, homatropine,
methscopolamine, homatropine methylbromide, methantheline,
cyclopentolate, tropicamide, propantheline, anisotropine,
dicyclomine, eucatropine and the like.
[0057] Mydriatics such as, atropine, cyclopentolate, homatropine,
scopolamine, tropicamide, eucatropine, hydroxyamphetamine and the
like. Psychic energizers such as 3-(2-aminopropy)indole,
3-(2-aminobutyl)indole and the like.
[0058] Antidepressant drugs such as, isocarboxazid, phenelzine,
tranylcypromine, imipramine, amitriptyline, trimipramine, doxepin,
desipramine, nortriptyline, protriptyline, amoxapine, maprotiline,
trazodone and the like. Anti-diabetics such as, insulin, and
anticancer drugs such as, tamoxifen, methotrexate and the like.
Anorectic drugs such as, dextroamphetamine, methamphetamine,
phenylpropanolamine, fenfluramine, diethylpropion, mazindol,
phentermine and the like. Anti-malarials such as, the
4-aminoquinolines, alphaaminoquinolines, chloroquine, pyrimethamine
and the like. Anti-ulcerative agents such as, misoprostol,
omeprazole, enprostil and the like. Antiulcer agents such as,
allantoin, aldioxa, alcloxa, N-methylscopolamine methylsuflate and
the like. Antidiabetics such as insulin and the like.
[0059] For use with vaccines, one or more antigens, such as,
whole-organism, natural, attenuated, heat-killed,
chemically-inactivated, synthetic, peptides and even T cell
epitopes (e.g., GADE, DAGE, MAGE, etc.) peptides and the like.
[0060] The drugs mentioned above may be used in combination as
required. Moreover, the above drugs may be used either in the free
form or, if capable of forming salts, in the form of a salt with a
suitable acid or base. If the drugs have a carboxyl group, their
esters may be employed. Acids may be an organic acid, for example,
methanesulfonic acid, lactic acid, tartaric acid, fumaric acid,
maleic acid, acetic acid, or an inorganic acid, for example,
hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric
acid. The base may be an organic base, for example, ammonia,
triethylamine, or an inorganic base, for example, sodium hydroxide
or potassium hydroxide. The esters mentioned above may be alkyl
esters, aryl esters, aralkyl esters and the like.
[0061] The present invention may be implanted into materials that
include sutures, tubes, sheets, adhesion prevention devices, wound
healing products, tissue healing agents and other tissue or cell
growth promoters that further enhance the effectiveness of tissue
regeneration. In addition, a voltage or current may be applied
directly to the present invention at the repair, implant,
transplant or reconstruction site. Polymers or other molecules with
piezoelectric or electrically conducting properties may also be
incorporated into the present invention. Several electroactive
polymers exist including piezoelectric (e.g., polyvinylidene
fluoride) and electrically conducting materials (e.g., polypyrrole
(PP), and polythiophene). Since piezoelectric materials depend on
small mechanical deformations to produce transient surface charges,
the level and duration of focused stimulation cannot be controlled.
In contrast, electrically conducting polymers readily permit
external control over both the level and duration of stimulation.
Thus strategies designed to enhance the regeneration of a
responsive cell might employ electrically conducting polymers. For
diagnostic purposes, the present invention may be incorporated not
only with molecules containing active species but also with one or
more detectable agents or molecules that allows for the diagnosis,
monitoring, and/or prophylactic measures. Examples of suitable
detectable agents include dyes, labels, metals, detection devices,
and electronic chips.
[0062] The extended release microencapsulated active agents may be
formed into compositions suitable for injectable use with the
Vitelline protein B additive to encapsulate the active followed by
dispersion in, e.g., sterile aqueous solutions and/or dispersions;
formulations including sesame oil, peanut oil and/or aqueous
propylene glycol; and/or sterile powders for the extemporaneous
preparation of sterile injectable solutions and/or dispersions. In
all cases the form must be sterile and/or must be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and/or storage and/or must be preserved
against the contaminating action of microorganisms, such as
bacteria and/or fungi.
[0063] Solutions of the active compounds as free base and/or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and/or mixtures thereof and/or in oils. Under ordinary
conditions of storage and/or use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0064] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle that contains the basic
dispersion medium and/or the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and/or freeze-drying techniques
that yield a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof The preparation of more, and/or highly, concentrated
solutions for direct injection is also contemplated, where the use
of DMSO as solvent is envisioned to result in extremely rapid
penetration, delivering high concentrations of the active agents to
a small tumor area.
[0065] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and/or in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and/or the
like may also be employed.
[0066] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary
and/or the liquid diluent first rendered isotonic with sufficient
saline and/or glucose. These particular aqueous solutions are
especially suitable for intravenous, intramuscular, subcutaneous
and/or intraperitoneal administration. In this connection, sterile
aqueous media that may be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage could be dissolved in 1 ml of isotonic NaCl solution and/or
either added to 1000 ml of hypodermoclysis fluid and/or injected at
the proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and/or
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0067] In addition to the compounds formulated for parenteral
administration, such as intravenous and/or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets
and/or other solids for oral administration; liposomal
formulations; time release capsules; and/or any other form
currently used, including cremes.
[0068] One may also use nasal solutions and/or sprays, aerosols
and/or inhalants in the present invention. Nasal solutions are
usually aqueous solutions designed to be administered to the nasal
passages in drops and/or sprays. Nasal solutions are prepared so
that they are similar in many respects to nasal secretions, so that
normal ciliary action is maintained. Thus, the aqueous nasal
solutions usually are isotonic and/or slightly buffered to maintain
a pH of 5.5 to 6.5. In addition, antimicrobial preservatives,
similar to those used in ophthalmic preparations, and/or
appropriate drug stabilizers, if required, may be included in the
formulation.
[0069] Additional formulations that are suitable for other modes of
administration include vaginal suppositories and/or suppositories.
A rectal suppository may also be used. Suppositories are solid
dosage forms of various weights and/or shapes, usually medicated,
for insertion into the rectum, vagina and/or the urethra. After
insertion, suppositories soften, melt and/or dissolve in the cavity
fluids. In general, for suppositories, traditional binders and/or
carriers may include, for example, polyalkylene glycols and/or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1%-2%.
[0070] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and/or the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations and/or powders. In certain defined embodiments, oral
pharmaceutical compositions will comprise an inert diluent and/or
assimilable edible carrier, and/or they may be enclosed in hard
and/or soft shell gelatin capsule, and/or they may be compressed
into tablets, and/or they may be incorporated directly with the
food of the diet. For oral therapeutic administration, the active
compounds may be incorporated with excipients and/or used in the
form of ingestible tablets, buccal tables, troches, capsules,
elixirs, suspensions, syrups, wafers, and/or the like. Such
compositions and/or preparations should contain at least 0.1% of
active compound. The percentage of the compositions and/or
preparations may, of course, be varied and/or may conveniently be
between about 2 to about 75% of the weight of the unit, and/or
preferably between 25-60%. The amount of active compounds in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0071] The tablets, troches, pills, capsules and/or the like may
also contain the following: a binder, as gum tragacanth, acacia,
cornstarch, and/or gelatin; excipients, such as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic acid and/or the like; a lubricant, such as
magnesium stearate; and/or a sweetening agent, such as sucrose,
lactose and/or saccharin may be added and/or a flavoring agent,
such as peppermint, oil of wintergreen, and/or cherry flavoring.
When the dosage unit form is a capsule, it may contain, in addition
to materials of the above type, a liquid carrier. Various other
materials may be present as coatings and/or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills,
and/or capsules may be coated with shellac, sugar and/or both. A
syrup of elixir may contain the active compounds sucrose as a
sweetening agent methyl and/or propylparabens as preservatives, a
dye and/or flavoring, such as cherry and/or orange flavor.
[0072] The mixed release pharmaceutical formulations disclosed
herein may be administered, e.g., parenterally, intraperitoneally,
intraspinally, intravenously, intramuscularly, intravaginally,
subcutaneously, or intracerebrally. Dispersions may be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms. The proper fluidity may be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms may be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, sodium chloride, or
polyalcohols such as mannitol and sorbitol, in the composition.
Prolonged absorption of the injectable compositions may be brought
about by including in the composition an agent that delays
absorption, for example, aluminum monostearate or gelatin.
[0073] Sterile injectable solutions may be prepared by
incorporating the therapeutic compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
therapeutic compound into a sterile carrier that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the methods of
preparation may include vacuum drying, spray drying, spray freezing
and freeze-drying that yields a powder of the active ingredient
(i.e., the therapeutic compound) plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0074] The bioactive may be orally administered, for example, with
an inert diluent or an assimilable edible carrier. The therapeutic
compound and other ingredients may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the therapeutic compound may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers
and the like. The percentage of the therapeutic compound in the
compositions and preparations may, of course, be varied as will be
known to the skilled artisan. The amount of the therapeutic
compound in such therapeutically useful compositions is such that a
suitable dosage will be obtained.
[0075] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit containing a predetermined
quantity of therapeutic compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (i) the unique
characteristics of the therapeutic compound and the particular
therapeutic effect to be achieved; and/or (ii) the limitations
inherent in the art of compounding such a therapeutic compound for
the treatment of a selected condition in a subject.
[0076] Vitteline protein B (vpB) is one of three proteins used by
parasitic worms to produce an encapsulant to protect the developing
embryo. The protein eggshell is formed predominantly of vpB through
the use of protein precursors which become crosslinked during the
maturation process through the use of dihydroxphenylalanine
residues. VpA and vpC, found in small quantities in the eggshell,
are believed to be involved in deposition and spreading of vpB. As
an immature, uncured protein, vpB demonstrates adhesive properties;
as a fully cured protein it serves as a novel sealant which is
resistant to acids, bases, heat, light, dessication, and
proteolysis. In addition the shell material is biocompatible and
non-antigenic. It has been found that vpB's resistance to
proteolysis makes it an ideal additive for microcapsules and micro-
and nanoparticles, extending particle life when introduced into an
animal. The vpB protein may also be used as a protein adhesive in
aqueous environments.
[0077] Vitelline protein B is a member of a family of proteins with
variable sequences, however, the amino acid composition is fixed.
The present inventors cloned and sequenced two members of the vpB
gene family and find their amino acid sequence to vary only within
the central 33% of the gene sequence. As shown in FIG. 1, the
sequence varies, the amino acid composition of the two vpBs (vpB1
and vpB2) is very similar; furthermore their amino acid composition
is nearly identical to that of the eggshell itself. As shown here
the amino terminal (N) ends of the proteins (approximately 1/3) and
the carboxy terminal (C) ends of the proteins (1/3) are identical
when comparing the two amino acid sequences. However the central
one third of the two proteins diverge significantly at the amino
acid level (35%). This shuffling of amino acids still preserves the
amino acid composition and the functionality of these
materials.
[0078] Table 1 summarizes the conservation of the amino acid
composition of two VpB proteins.
1 Purified vpB AMINO ACID PROTEIN VpB1 cDNA* VpB2 cDNA* ASX 140 +
4.3 158.1 134.0 THR 18 + 3.1 15.8 15.8 SER 52 + 4.8 55.3 51.4 GLX
83 + 3.7 83.0 75.1 PRO 16 + 0.9 11.9 11.9 GLY 165 + 4.6 150.2 154.2
ALA 69 + 2.4 71.1 71.1 CYS/2 0 0 0 VAL 9 + 2.1 7.9 4.0 MET 23 + 4.2
23.7 23.7 ILE 5 + 1.0 4.0 0 LEU 38 + 1.4 35.6 35.6 DOPA 106 + 9.8
TYR 21 + 5.0 134.4 138.3 PHE 38 + 3.1 35.6 35.6 HIS 45 + 3.7 33.3
55.3 LYS 120 + 6.5 114.6 126.5 ARG 60 + 3.2 63.2 67.2 TRP 0 0 0
*values represent mature vpB exclusive of signal sequence.
[0079] As such, the composition rather than the sequence of these
proteins is responsible for the properties of 1) resistance to
proteolysis and 2) adhesiveness.
[0080] Polymers containing catecholic groups are widespread in
nature and perform diverse functions. In microbial systems the
polymers consist of short peptides whose function is to sequester
ferric iron from the environment (Raymond and Carrano 1979, Ong et
al 1979). In marine invertebrates such as mussels and tunicates the
catecholic polymers are proteins modified by the presence of
dihydroxyphenylalanine (DOPA) residues whose function is underwater
sealant and adhesive (Waite et al 1985, Waite 1986). The synthetic
counterparts of these polymers have found widespread application in
industry as semiconductors (Jaegfeldt et al 1983, Lau and Miller
1983), metal chelators (Pecoraro et al 1981), electrocatalysts
(Degrand 1985) and adhesives (Pizzi 1985) to name a few. Additional
interest in DOPA compounds has been generated by the recent finding
that DOPA derivatives may act as redox cofactors at the active site
in amine oxidases (Janes et al 1990). The freshwater trematode,
Fasciola hepatica, produces a catecholic protein polymer which
functions as a structural protein in egg microencapsulation. The
polymer is crosslinked and quinone-tanned to produce a sclerotized
egg case with extraordinary properties.
[0081] Faciola hepatica is a digenetic trematode which encapsulates
its eggs in a proteinaceous shell. The shells are a prototypical
microencapsulating system that protects Fasciola eggs from the host
natural defenses while allowing uptake of essential nutrients and
release of metabolic products. The long-term stability of this
natural composite material results from crosslinks formed by
quinone tanning or sclerotization of specialized eggshell proteins
in which tyrosine residues have been post-translationally modified
to 3,4-dihydroxyphenylalanine (DOPA). The present inventors
purified and characterized the three major protein components of
the shell, vitelline proteins A, B and C (vpA, vpB, and vpC). In
addition, cDNAs encoding two variants of the major eggshell
component have been sequenced and expressed as recombinant protein.
The proteins encoded consist of highly degenerate repeats of a
hexapeptide enriched in glycine and containing clusters of basic
amino acid residues as well as clusters of acidic residues.
[0082] The vitelline proteins, natural encapsulating agents, offer
several advantages over materials in current use, such as synthetic
polymers and gelatin. (1) As recombinant proteins they can be
obtained in bulk with uniform and defined characteristics. (2) The
protein compositions can be genetically engineered to vary the
density of tyrosine/DOPA residues, or introduce cysteine residues
and so control the cross-linking characteristics and the resultant
porosity and stability of microcapsules. (3) The cross-linking
agent is already integrated into the protein and need be only
oxidized, either spontaneously or with easily separable reagents,
to initiate curing. (4) the vitelline proteins are poorly
antigenic. (5) Though durable, cured protein is (ultimately)
biodegradable and non-toxic.
[0083] Synthetic microencapsulation. Proteins have enjoyed
extensive use as encapsulating agents and so there exist numerous
protocols which may be applied to vitelline protein
microencapsulation. In essence, all approaches involve the
induction of a phase separation in a mixture of core material and
encapsulating agent such that the core is efficiently engulfed by
the encapsulating agent, usually followed by stabilization of the
microcapsule walls by low molecular weight cross-linking agents
such as glutaraldehyde. Phase separations are commonly induced by
i) manipulation of temperature, pH, salt or alcohol concentrations;
ii) addition of incompatible polymers; iii) liquid coacervate
formation; and iv) congealing or denaturation in oil emulsions.
Others approaches are suggested by the biochemical properties of
vpB, such as interfacial polymerization at liquid/liquid interfaces
or the stabilization of liposomes encased in vitelline protein by
covalent cross-linking.
[0084] The present inventors have studied the mechanism by which
the worm microencapsulates in order to mimic that process in vitro.
The worm carries out the process of microencapsulation by applying
a film of eggshell precursor protein to the surface of a
lipoprotein layer or interface formed in the lumen of Mehlis'
gland, the site of eggshell assembly. A catechol oxidase activity
oxidizes the DOPA residues of the eggshell precursor to
DOPAquinone. The highly reactive DOPAquinone is postulated to
spontaneously form a number of types of chemical cross-links
although in this system lysine or histidine appear to be the
primary nucleophiles participating in cross-linking. Faithfully
mimicking the worm's approach will therefore involve the use of
liposome encapsulation followed by spreading of recombinant
eggshell protein on the outer surface of the liposome. A
commercially available mushroom tyrosinase may then be employed to
crosslink the shell precursor proteins in vitro and to cure the
microcapsule.
[0085] Alternatively, coacervation methods based on gelatin
microcapsulation (simple coacervation) or gelatin/acacia
microencapsulation (complex coacervation) may be used. Either
approach eliminates the need to first encapsulate the core material
in a liposome prior to encapsulation with vitelline protein.
[0086] One or more of vitelline proteins vpA, vpB, and vpC may be
contacted with an agent to be microencapsulated under conditions
suitable for microencapsulation. The agent can be a peptide,
protein, nucleic acid, pharmaceutical, or other organic chemical
compound. The nucleic acid and protein sequence of vpB can be found
at the NCBI database, Accession No. M93024. Additionally, the
vitelline proteins may be modified by enzymatic or chemical
post-translational modification. One example of such a modification
is the hydroxylation of one or more tyrosine residues.
Additionally, fragments of the vitelline proteins can be used in
place of, or in combination with the full length naturally
occurring proteins.
[0087] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Purification and Characterization of Protein Components of
Shells
[0088] Documentation of Fasciola hepatica eggshell production has
historically included a quinone tanning process which involves
cross-linking of proteins containing catecholic groups (Smyth and
Clegg, 1959). However the nature of the substrates involved in the
cross-link and the structure of the cross-link itself have been
subjects for heated debate and novel investigative approaches.
Waite and Rice-Ficht demonstrated the abundance of
dihydroxyphenylalanine (DOPA) in the shell producing glands of the
worm (vitellaria) as well as in the shells themselves using a DOPA
specific reagent recognizing only monosubstituted 1,2 benzenediols
and producing a bright red chromophore. The results were highly
suggestive that DOPA is at least one of the major substrates for
oxidation and protein crosslinking. Several DOPA-containing protein
precursors of the shell were subsequently purified and
characterized. The three major DOPA-containing proteins of the worm
are rich in glycine and DOPA and compose three major size classes
of 70 kDa, 31 kDa and 17 kDa. Each protein also has a distinctive
amino acid composition with the 70 kDa protein rich in ASX and ARG,
the 31 kDa protein rich in LYS and ASX the 17 kDa protein rich in
HIS.
Example 2
Characterization of 31 kDa vpB Protein
[0089] The 31 kDa protein (vitelline protein B, vpB) has been
purified and characterized in detail (Waite and Rice-Ficht 1987;
Waite and Rice-Ficht 1992, appendix 3; and Rice-Ficht and Waite
1992, appendix 2). Sequence analysis of tryptic peptides derived
from this protein constituted the first direct demonstration that
DOPA residues were in fact a component of the peptide backbone and
were likely formed through post-translational modification of
tyrosine residues of the precursor protein; this parallels
observations in the adhesive proteins of the marine mussel, Mytilis
edulis, (Waite and Tanzer 1980) but is quite distinct from other
mechanisms documented for quinone tanning such as that employed by
insects in the formation of cuticles and egg cases (Richards 1978).
The protein has an apparent molecular weight of 31,000, a pI of 7.4
and constitutes approximately 7% of the protein in adult Fasciola
hepatica. Eleven per cent of the amino acid residues of the protein
are DOPA residues which `disappear` during shell curing in vivo or
during treatment with mushroom polyphenol oxidase in vitro.
Example 3
Isolation of cDNA Encoding vpB Protein
[0090] Isolation of cDNAs encoding the 31 kDa protein was achieved
using a combination of antibody selection and hybridization with
degenerate oligonucleotides (Rice-Ficht and Waite 1992). The
proteins encoded are approximately 30,900 daltons and bear a
striking homology with the amino acid composition of the purified
protein. Sequencing of cDNAs has revealed the presence of at least
two distinct mRNAs encoding vitelline protein B1 (vpB1) and
vitelline protein B2 (vpB2) which are markedly different in amino
acid sequence (33% variation) but quite similar in amino acid
compositions. Southern blot analysis also indicates the presence of
at least six gene copies for the 31 kDA protein when vpB1 is
employed as a hybridization probe under stringent conditions. These
results led to a re-examination of the apparently homogeneous 31
kDa protein and revealed microheterogeneity and the presence of a
family of proteins.
[0091] The electrophoretic microheterogeneity is attributed to i)
varying degrees of post-translational modification (TYR to DOPA)
and ii) the presence of mRNAs varying in primary sequence (Waite
and Rice-Ficht 1992, appendix 3). Fractionation of the purified
protein was achieved through C-8 reversed phase HPLC; and the
fractions across the vpB peak were subjected to amino acid
analysis. All amino acids were constant across the peak with the
exception of tyrosine and DOPA. DOPA residues were high on the
leading edge of the peak and declined with increasing acetonitrile
concentration; the converse was true of tyrosine residues
suggesting a precursor-product relationship. N-terminal sequence
analysis of protein fractions across the peak indicates that all
proteins share the same N-terminus. This data taken alone might
indicate that the heterogeneity was due solely to
post-translational modification. However, extensive sequencing of
tryptic peptides has shown the presence of peptides unique to the
vpB1 mRNA and to the vpB2 mRNA.
[0092] Both vpB1 and vpB2 contain the N-terminal peptide sequence
defined through vpB peptide analysis. Southern hybridization was
carried out on duplicate filters using either the vpB1 gene as
probe or an oligonucleotide which represents the N-terminus of all
vpB proteins (data not shown). There appear to be 6-7 structural
genes for this family of proteins while the N-terminal sequence is
associated with only one copy. However, the data derived from
sequencing vpB1 and vpB2 indicates that at least two different
transcripts carry this N-terminal sequence. The possibilities for
this dichotomy are many but the likeliest explanations are cis,
trans or possibly differential RNA splicing are placing the single
N-terminal coding sequence on multiple transcripts; this is being
pursued. One approach to studying the number of transcripts which
bear the N-terminal sequence is to employ PCR using RNA as
substrate.
Example 4
Characterization of 17 kDa vpC Protein
[0093] Analysis of the 17 kDa eggshell protein (vitelline protein
C, vpC) has revealed a similar heterogeneity with the presence of
at least four distinct electrophoretic types. Apparent molecular
weights range from 16,000 to 18,500 while pIs under denaturing
conditions indicate the presence of only two species of 6.89 and
6.99. A single amino terminal peptide sequence is detectable and
has been employed as a tool for gene isolation using degenerate
oligomers. The amino acid composition of vpC is remarkable in that
DOPA (20%), histidine (20%), and glycine (41-42%) comprise 80% of
the amino acid residues in all variants of the family. The bulk of
the molecule is composed of a (GLY-X)n repeat motif in which X is
SER, DOPA or HIS. The only feature which this protein shares with
the 31 kDa protein is the presence of DOPA residues. The four or
more variants observed in the vpC family do not arise as a result
allelic differences between individuals since a survey of six
morphologically different worms indicates the presence of all vpC
variants in each individual (Waite and Rice-Ficht 1989).
Example 5
Temporal and Spatial Expression of RNA and Proteins
[0094] Through the use of antisera specific for the 31 kDa protein
and through in situ hybridization it has been possible to study the
localization of expression of the major DOPA protein. The use of a
rabbit antisera raised against the purified vpB in tissue
localization studies has indicated a high level of the protein
within vitelline cells localized to the vitelline glands at the
worm periphery (Rice-Ficht 1992); reactivity of antisera
corresponds to the vitelline globules of mature vitelline cells
(Rice-Ficht and Waite, 1992; appendix 2) and is evident before
vitelline cells pass into the vitelline reservoir for delivery to
Mehlis' gland, the site of shell assembly. The 31 kDa protein is
synthesized and stored in vitelline granules of vitelline cells
still residing in the vitelline gland follicle.
[0095] In situ hybridization using the carboxyterminal 40% of the
vpB1 gene agrees with the antibody localization data (Rice-Ficht
and Waite 1992). Transcripts encoding vpB1 are most abundant in the
earliest form of mature vitelline cell residing in the vitelline
gland. The protein apparently enters a pathway for regulated
secretion and is stockpiled in globules of the vitelline cell
cytoplasm. A putative chemical signal elaborated by Mehlis' gland
triggers release of the globules and the initiation of capsule
assembly.
Example 6
Putative Models for Shell Assembly
[0096] Based on a body of histological and histochemical data
reviewed by Smyth and Clegg (1959), metabolic labeling studies
(reviewed by Rice-Ficht 1992) and the current biochemical analysis
of eggshell proteins, a biological and biochemical model of shell
assembly are proposed. In these models all three shell precursor
proteins are translated in the extensive GER of the early vitelline
cells and stockpiled in globules prior to the time that the cells
leave the follicle. Nascent protein precursors are modified by a
putative protein-specific tyrosyl hydroxylase to produce Dopa
residues in the protein backbone. The polyphenolic shell precursors
(Smyth 1954; Waite and Rice-Ficht 1989) and polyphenol oxidase
(Smyth 1954) are transported to the ootype and packed away in
vitelline cell globules. As vitelline cells congregate in the
ootype along with one fertilized ovum, Mehlis' gland forms a
lipoprotein membrane (Clegg 1965) around the vitelline cell/egg
mass which is followed by release of globular material from the
vitelline cells through regulated secretion. As the globule
contents spread on the lipoprotein membrane, catechol oxidase
becomes activated and oxidation and crosslinking ensues. Dopa
residues are abundant in newly formed shells and shells of the
proximal uterus. As the shells pass through the uterus, Dopa
residues are consumed in the crosslinking reactions (as ascertained
through DOPA-specific staining; Waite and Rice-Ficht 1989) and the
shells are completely `cured` prior to extrusion.
[0097] Some clues to the role of individual proteins in shell
formation have presented themselves. VpB is the major shell
component by weight and is present in molar quantities
approximately 30 times that of vpA or vpC. Of the three proteins
studied only one, VpC, has substantial homology with any published
protein sequence; vpC shows strong homology with the his-rich
domain of high molecular weight kininogen I whose function is to
bind to negatively charged surfaces and accelerate binding of other
blood clotting factors to that surface (Kitamura et al 1983). Based
on this homology, it is postulated that vpC is the first of the
shell proteins to associate with the lipoprotein layer,
facilitating binding and dispersion of vpB and vpA during shell
formation.
Example 7
Characterization of the 70 kDa DOPA Containing Protein of the
Eggshell
[0098] The 70 kDa protein (vitelline protein A, vpA) is the last of
the three major DOPA containing protein precursors of the eggshell
remaining to be characterized. Preliminary amino acid analysis has
indicated a high ASN/ASP (21%), GLY (11%) and ARG (8%) content with
4% DOPA residues and a substantial amount of unmodified tyrosine.
One of the future goals of the project is to compare in vitro
modification of the vpA, vpB and vpC with various polyphenol
oxidases in order to probe the factors governing modification of
individual tyrosine residues. Further characterization will include
analysis for microheterogeneity and limited peptide sequencing.
Example 8
Purification and Characterization of the F.hepatica Catechol
Oxidase
[0099] Present evidence suggests that catecholoxidase activity is
present in mature vitelline cells as well as eggshells of F.
gigantica (Nellaiappan & Ramalingam, 1980), S. mansoni (Seed
and Bennett 1980), S. japonicum (Wang et al 1986), and Parapleurus
sauridae (Nellaiappan and Ramalingam 1980), where it presumably
catalyzes the oxidation and subsequent polymerization of the
eggshell precursor proteins to a quinone-tanned material. These
studies, however, do little more than confirm the presence of
enzyme activity.
[0100] F. hepatica from freshly condemned bovine livers will be
obtained from the slaughterhouse in Sealy, Tex. and transported to
the laboratory on dry ice. Extraction of soluble enzyme activity
from the vitellaria is complicated by at least three factors, a)
vitellaria interdigitate with the digestive diverticulum hence any
extraction is likely to contain enzyme with some contaminating
proteases, b) like zymogens, the catecholoxidases of insects, frog
skin and mussels (Waite 1985) are stockpiled in latent form prior
to secretion, and c) the catecholoxidases implicated in
quinone-tanning reportedly have odd solubility requirements and
commonly become co-crosslinked to their substrates.
[0101] In order to address these concerns, enzyme extractions will
be done using buffers with a broad spectrum of protease inhibitors,
e.g., 1 M phenylmethylsulfonylfluoride, 10 mM N-ethylmaleimide, 25
mM ethylenediaminetetraacetic acid (EDTA). Good activation of other
latent catecholoxidases has been achieved with trypsin and,
especially, chymotrypsin treatment. There is no fait accompli
recipe for solubilizing catecholoxidase activity from vitelline
cells. Wang, et al., (1986) extracted the enzyme from S. japonicum
with unbuffered 0.25 mM sucrose, others have utilized unbuffered
0.25 M sucrose (Nellaiappan and Ramalingam, 1980) and 0.01 M sodium
phosphate at pH 7.2 (Thangaraj et al 1986) to solubilize enzyme
from monogeneans. Unfortunately, Seed et al., (1978) and Mansour
(1958) report a conflicting conclusion that catecholoxidase
activity in F. hepatica and S. mansoni is not soluble in 0.1 M
potassium phosphate at pH 6.8. Clearly, many of these studies are
of marginal value and should be redone. Some precautions to improve
yields might be to use 1 M NaCl in the extraction buffer. This
improves recovery of enzyme from quinone-tanned byssal threads
(Waite 1986). Addition of potassium cyanide or salicylaldoxime to
crude extracts may reduce premature oxidations by the enzyme, and
use of borate as the extraction buffer will complex (hence
inactivate) intrinsic substrates such as DOPA-containing
proteins.
Example 9
Purification of Extracted Enzyme
[0102] Purification of the extracted enzyme will be attempted by
ion exchange chromatography and gel filtration. The assay of
catecholoxidase activity will be done using the assay based on the
formation of quinone-proline adducts (Rzepecli and Waite 1989) and
by measurement of O.sub.2 consumption using an oxygen electrode
(Duckworth & Coleman 1970). The formation of quinone-proline
adducts is linear with time and results in a deep purple
chromophore (molar E.sub.390=8300 cm.sup.-1). Following the
purification of the enzyme, a physical characterization of the
enzyme will include molecular weight determination by gel
filtration using the appropriate range of standards, subunit
molecular weight determination by SDS PAGE, and prosthetic metal
determination by atomic absorption spectroscopy. All
polyphenoloxidases known to date are copper proteins. Amino acid
composition will be performed following protein hydrolysis
according to Tsugita, et al, (1987). The N-terminus of purified
subunits will be sequenced by Edman gas phase methodology (Ozols
1986). An alternate approach will be an enzyme isolation from newly
formed eggshells, purified from the Fasciola uterus; complete
digestion of the shells with highly purified enzymes specific for
DOPA residues (from the marine organism Alteromonas, see below in
cross-link analysis) may release the catecholoxidase. Since the
enzyme is known to be active even in completely cured eggshells
this approach may yield some positive results.
[0103] Enzyme-substrate kinetics will be a particular focus of
these studies. A comparison of the Michaelis-Menten behavior of the
vitelline and eggshell enzyme activities with various synthetic
mono- and diphenols and O.sub.2 will be performed. In addition,
DOPA-containing peptides derived from protease digests of vitelline
proteins B and C will be employed as substrate. Effect of
inhibitors, pH, and temperature will be determined using the best
substrate (highest Vmax/Km) and heat denatured-controls. Use of
intact proteins as substrates is impractical due to the
insolubility of these without borate at physiological pH (Waite and
Rice-Ficht 1987).
[0104] In structures undergoing quinone tanning such as nascent
eggshells, it is not clear whether assembly occurs in a liquid
crystal or solid-state (Bouligand, 1985; Waite, 1985). The amount
of enzyme present would certainly have a bearing on which of the
states existed. In the liquid crystal, the enzyme would presumably
have more freedom to move from one crystalline precursor to another
in the course of introducing crosslinks. In the solid state, in
contrast, enzyme activity would be severely localized e.g., as in
an eggbox and much more of it would be required relative to the
liquid crystal model to effect crosslinking. Information about the
concentration of enzyme in the eggshells could thus indirectly at
least invalidate the liquid crystal or solid state models. If the
specific activity of catecholoxidase purified from vitelline cells
can be calculated, then in principle, the total amount of active
enzyme per mg eggshell protein can be estimated according to Segel
(1976). Recall that catecholoxidase activity persists in eggshells
even after release of the egg from the host (Smyth and Clegg 1959).
The major assumption will be that the specific activity of the
enzyme is not drastically altered once it becomes part of the
eggshell. Total protein in the eggshell will be quantitated by
amino acid analysis following hydrolysis. A corroborative estimate
of the amount of catecholoxidase in eggshells (based on the mol %
Cu detected in purified catecholoxidase) could be attempted by
doing atomic absorption measurements on hydrolyzed eggshells. Here
the assumption would be that all eggshell Cu is from the
catecholoxidase.
[0105] Numerous attempts to purify the trematode catechol oxidase
from freshly prepared worms applying techniques successfully
employed in M. edulis have been unsuccessful. Techniques which
successfully fractionate the enzyme often lead to its inactivation
before homogeneity can be achieved. A new purification scheme
involving the use of phase partition with Triton X 114 (Bordier
1981; Sanchez-Ferrer 1989) will next be employed to fractionate and
purify the trematode enzyme. The enzyme plays a pivotal role in
eggshell assembly and its purification is a priority of the
project. Initial characterization of the trematode enzyme in
relatively crude preparations indicates it to be present in
stoichiometric rather than enzymatic quantities with respect to the
shell precursor proteins vpA, vpB and vpC. This raises the distinct
possibility that the enzyme is not only catalytic but an integral
part of the shell architecture becoming crosslinked and immobilized
during shell assembly. This possibility is underscored by the fact
that the enzyme may be assayed in partially cured eggshells but
never isolated (data not shown). Histochemical evidence also
suggests that the enzyme is present in an inactive form even in
newly formed vitelline globules as an emulsion with the vpA, vpB
and vpC substrates. This provides the enzyme/substrate combination
in a premixed form prior to spreading and quinone tanning.
[0106] An alternate approach to enzyme purification by conventional
methods will be an affinity purification scheme; this approach
would utilize antibody directed against recombinant catechol
oxidase coupled to cyanogen bromide activated sepharose beads. This
approach is dependent upon isolating the gene first and producing
recombinant protein from that gene (objective IIIB). Affinity
purification using substrate covalently attached to beads (i.e. a
methyl catechol) might also be employed by a method analogous to
that used for the purification of the Schistosoma
glutathione-S-transferase via bead-bound glutathione (Smith and
Johnson 1988).
Example 10
Characterization of cDNAs Encoding the 17 kDa Proteins
[0107] cDNAs were isolated from lambda gt10 and lambda gt11 F.
hepatica libraries using hybridization and a degenerate
oligonucleotide probe. The N-terminal sequence of vpC was employed
to produce a 29-mer using inosine substitution at the most
degenerate positions in order to limit the number of
oligonucleotides in the mix (Ohtsuka et al 1985). The probe was
employed using tetramethylammonium chloride salts, a method which
provides a base composition-independent hybridization (Wood et al
1985). The hybridization can be controlled as a function of probe
length only, enhancing results when screening a complex library
with a pool of oligonucleotide probes. A number of cDNAs of 500-600
base pairs in length have been isolated and sequence is being
determined. Sequencing is proceeding through application of
asymmetric polymerase chain reaction (Gyllensten and Erlich 1988)
directly from the selected lambda library clones. Primers flanking
cDNA insertion sites in the vector are employed to amplify the DNA;
following a brief extraction procedure to remove primers and
nucleoside triphosphates the PCR product is subjected to sequencing
with standard dideoxy chain termination methods and T7 polymerase
(Sanger and Coulson 1975). This method which circumvents subcloning
and other low-efficiency, time consuming procedures is being
employed to examine the clones.
Example 11
Isolation and Characterization of cDNAs Encoding the 70 kDA DOPA
Protein
[0108] A similar approach may be used to isolate the vpB1 and vpB2
genes (Rice-Ficht and Waite 1992). Adult F. hepatica libraries have
been constructed in this laboratory and include cDNA libraries in
lambda gt10 and lambda gt11 and genomic libraries in lambda 2001
and lambda DASH; each has been used successfully for gene
isolation. In view of the low level antigenicity of vpB and vpC a
dual approach to gene isolation will be continued. Antibody will be
raised to the purified vpA and employed for cDNA isolation from the
lambda gt11 library (Davis and Young 1983). Limited peptide
sequencing will also be employed to define oligonucleotides for
library screening and gene isolation.(Wood et al 1985).
Example 12
Isolation of the Gene Encoding trematode Catechol Oxidase
[0109] Lambda gt11 libraries produce proteins as a fusion with the
116 kDa b-galactosidase gene which may easily interfere with proper
folding and activity of the cloned enzyme. Early attempts to carry
out plaque assays with Arnow's reagent (Arnow 1937) and 4-methyl
catechol were unsuccessful due to the solubility and instability of
the chromophore produced. A modification of this protocol (Rzepecki
and Waite 1989) producing a more intense color reaction was also
employed without success. An alternate approach using an antibody
(polyclonal) directed against the Mytilis edulis catechol oxidase
will be used to identify cloned trematode enzyme in an expression
library. Although catechol oxidases investigated to date from
various sources due not share significant nucleotide or amino acid
sequence homology, secondary structures of the enzymes may be
similar. Additionally, the trematode and mussel enzymes may be more
closely related in structure in view of the similarities in
substrates (protein-bound tyrosine) and processes in which they
participate.
Example 13
Selection of cDNAs Encoding Proteins Specific to Mehlis' Gland from
a Mehlis' Gland Specific Expression Library
[0110] The Mehlis' gland constitutes the biochemical "black box" of
eggshell formation; among the functions attributed to the gland are
i) initial entrapment of 30 vitelline cells and one ovum in a lipid
bilayer (Wharton 1983), ii) signaling the release of vitelline
globules from the vitelline cells and iii) activation of the
catechol oxidase enzyme. In an effort to identify additional
products and processes involved with shell manufacture and the
production of the shell material, a selection process will be
carried out to identify proteins manufactured only in Mehlis'
gland. One interesting observation in our laboratory involves the
finding that histochemically the uterine lining which interfaces
with Mehlis' gland contains a high concentration of membrane-bound
alkaline phosphatase. This is only one example of Mehlis' gland
specific products which may be isolated via the process outlined
here. We postulate that the presence of this enzyme may be
significant in relation to production of a phosphate buffering
system in the lumen of Mehlis' gland. pH may be especially
important in the spreading properties of histidine rich proteins
(i.e. vpC) since the imidazole pK is near pH 7.0 (Rice-Ficht
1992).
[0111] The production of a Mehlis' gland-specific cDNA library will
be carried out through the standard techniques first applied to the
manufacture of T cell-specific cDNA libraries via "subtraction
hybridization" (Hedrick et al 1984). For this procedure flash
frozen worms will be partially thawed and the Mehlis' gland area
from 50 worms excised and pooled; the anterior portion or "head" of
the worms will also be pooled. The anterior portions are assumed to
contain the standard housekeeping genes found in all cell types and
will be used to remove unwanted RNA from the Mehlis' gland
preparation prior to cDNA synthesis. RNA will be extracted from the
tissues as described (Rice-Ficht and Waite 1992) and a first strand
cDNA synthesized using the Mehlis' gland RNA as template. The cDNA
will be hybridized to a 100-fold excess of head RNA and applied to
hydroxyapatite. The cDNA not retained by the column should be
single-stranded and unique to Mehlis' gland; a second cDNA strand
will be synthesized and the cDNA introduced into lambda gt11 and
lambda gt10 for amplification. There are alternate protocols to
achieve the same objective including deletion enrichment (Lamar and
Palmer 1984) and a plus/minus screening for standard cDNA libraries
(Tedder et al 1988; Zurita et al 1987).
[0112] cDNAs of interest will be located using antibody prepared
against excised Mehlis' gland or through a plus/minus hybridization
technique (Tedder et al 1988). The later relies on screening
duplicate plaque lifts from a cDNA library with a radiolabeled cDNA
representing first the tissue of interest (i.e. Mehlis' gland) and
secondly a tissue containing background genes (i.e. the worm
`head`). Plaques screening positive with the gland probe and
negative with the head probe would be candidates for further study.
The Mehlis' gland specific nature of candidate sequences will be
determined through i) in situ hybridization of the cloned segment
to worm sections to verify its presence in Mehlis' gland, ii)
production of recombinant protein against which polyclonal and
monoclonal antibody would be raised for tissue localization studies
or iii) sequencing of the cDNA through polymerase chain reaction to
perform a preliminary search of the gene bank.
[0113] A limited success with the production of monoclonal antibody
against the shell precursor proteins prompted a multifaceted
approach to gene isolation. Additionally, monoclonal antibody is
often a poor reagent for clone selection from expression libraries
due to the fact that only a single epitope is recognized by the
antibody. The major utility of monoclonal antibody will be in
tissue localization of proteins and fine structure mapping at the
EM level.
Example 14
Analysis of the Metal Composition of Shells Isolated Directly from
the trematode Uterus
[0114] The suggestion has been made that various metals are
associated with trematode vitellaria. The observation of
"calcareous corpuscles" within the mature vitelline cells of
Schistosoma mansoni which are rich not only in calcium but in
phosphorus and magnesium as well (Shaw and Erasmus 1984) is one of
the strongest. Coupled with the fact that DOPA residues have
extraordinary binding constants for a number of metals we propose
to examine the metal content of the vitelline cells of F. hepatica
as well as purified eggshells for metal composition. Initial
studies will be carried out using electron probe microanalysis and
energy dispersive X-ray spectroscopy (Shaw and Erasmus 1984).
Element quantities as small as 10-18 g in subcellular structures
may be probed (Hall 1979). Cryosections of adult worms will be
employed to minimize changes which might occur with fixation. Both
vitelline cells and eggshells within the worm will be examined in
cryosection although additional analysis of shells carefully
purified from contents in the absence of metal chelating agents
will be performed. The electron probe microanalysis technique is
preferred initially over more precise physical methods because it
requires less material and should serve well for initial survey
purposes.
Example 15
Study of Metabolism and Deposition of Shell Protein Components
[0115] Tissue culture techniques in trematodes have been largely
unsuccessful in the study of eggshell formation. Although many
aspects of worm metabolism are apparently preserved under standard
culture conditions shell deposition becomes markedly aberrant after
only minutes to hours in culture (Clegg 1965; Smyth and Clegg
1959); free vitelline cells and free globules of shell precursor
material rapidly appear in the proximal uterus during culture, a
phenomenon which is not observed in worms immediately after removal
from the host. And, although egglaying by trematodes continues for
hours to days after introduction into culture, the process relies
on preformed RNA and protein since RNA production for shell
precursor ceases with introduction to culture (Reis et al 1989).
The present inventors conducted culture of trematodes in fertilized
chicken eggs as recently reported by Fried (1989) for other genuses
of flatworm and found that egglaying proceeds in an apparently
normal fashion (i.e. a lack of vitelline cells or free vitelline
globules in the uterus) for extended periods of time (tested up to
ten days). Analysis of culture worms for the presence of mRNA
complementary to shell precursor protein genes is underway. Since
F. hepatica is auxotrophic for a remarkable number of compounds
including purines, pyrimidines, sterols, fatty acids, and a number
of amino acids (Kurelec 1972) we anticipate the ability to
successfully label macromolecules even in amniotic fluid of the
avian egg. Successful isotopic labeling of shell precursor proteins
will enable a range of analyses concerning shell crosslinking and
shell architecture to be performed.
Example 16
Analysis of Eggshell Cross-Links Using Solid State NMR
[0116] Cross-links in insect cuticle have been successfully probed
employing solid state .sup.13C and .sup.15N NMR (Schaeffer, et al,
1987). In this procedure tobacco hornworm larvae were injected with
either 13C (ring labeled) dopamine to label catechols or with
.sup.15N histidine; NMR analysis of intact cuticle revealed the
presence of covalent linkages between protein bound histidine and
catecholamine dopamine. Similar analyses will be carried out in the
trematode system labeling worms with .sup.13C tyrosine as DOPA
precursor, .sup.15N lysine and .sup.15N histidine to probe possible
crosslinks between vpB, rich in DOPA and lysine, and vpC, rich in
DOPA and histidine.
Example 17
Analysis of Eggshell Cross-Links Using Proteolysis with DOPA
Specific Enzymes
[0117] Another approach to characterize crosslinks would rely on
the use of Dopa-protein digesting proteases recently described from
the marine Alteromonas species (Dohmoto and Miyachi 1991). These
enzymes completely digested byssyl thread and were found to contain
at least two proteases that preferentially cleaved the peptide bond
next to DOPA. For this study we propose to digest eggshells
harvested and cleaned from the uteri of F. hepatica with a crude
Alteromonas protease (buffer 0.1 M Tris-ascorbate pH 7.5) for as
long as necessary to render the eggshells completely soluble. When
this has been accomplished, residual material will be removed by
centrifugation, and the supernatant lyophilized, redissolved in 5%
acetic acid and separated on C-18 reversed phase HPLC. Fractions
containing aromatics will be examined by amino acid analysis, UV
spectrophotometry and, if encouraging, by mass spectrometry.
Example 18
Analysis of Eggshell Cross-Links Using Specific Labeling Through
Nucleophilic Addition of .sup.14C Glycine Ethylester
[0118] The greatest limitation in working with quinone-tanned
proteins is that they typically resist every treatment short of
complete hydrolysis. For this reason entirely, it is expedient to
work with pretanned precursors. The present inventors adopted a
labeling protocol developed by Simon and Green (1988) for following
the course of cross-linking in involucrin, a major protein of the
epidermis. Like the vpB and vpC, involucrin (mol. wt. 100 kDa)
consists of a degenerate series of repeating consensus decapeptides
that are rich in glutamine (Eckert and Green 1986). It was recently
observed that involucrin in terminally differentiated epidermal
cells is associated with tissue transglutaminase, a crosslinking
enzyme.
[0119] An iso-peptide bond is formed from peptidyl-glutamine and
-lysine. In undertaking to determine which glutamines are targeted
for cross-linking, Green and co-workers opted for an approach that
discarded the physical problems associated with crosslinking (such
as insolubility and intractability). This was done by overwhelming
the natural amine donor peptidyl lysine with 14C glycine
ethylester. The 14C glycine ethyl ester modified protein could be
i) visualized on gels, ii) isolated by HPLC and iii) digested into
a family of radioactive and nonradioactive peptides. These of
course can be easily purified and sequenced. In the case of
involucrin, the results obtained were most intriguing and totally
unexpected. Of the 39 or so consensus repeats in intact involucrin,
glutamines in only two of the repeats were consistently labeled
(Simon and Green, 1988).
[0120] VpB and vpC are more degenerate in terms of repeats but they
can be analogously treated with mushroom tyrosinase at pH 8.0
(phosphate buffered saline) to form messy cross-linked aggregates
of high molecular weight (Waite and Rice-Ficht, 1987). Lysines are
implicated in the cross-linking by two lines of evidence including
i) lysine levels decrease with DOPA as the course of the oxidation
proceeds, and ii) vpB is less trypsin-labile following oxidation
(Waite and Rice-Ficht 1987; unpublished observation). If lysine in
vpB were overwhelmed with .sup.14C glycine ethyl ester (10 uCi, 2
mM), then in principle this protein (200 ug/50 ul) too should
become increasingly labeled without necessarily becoming insoluble
or resistant to trypsin. A cautionary note is advisable since other
cross-links are possible. Even so, if crosslinking is limited the
vpB would be digested with trypsin and tryptic peptides could be
screened via C-8 reversed phase HPLC (Waite et al 1985) and liquid
scintillation for `hot` peaks. Characterization of hot peaks by
amino acid analysis and microsequencing will help identify the
position and number of dopaquinones targetted. There are at least
four potential addition sites for glycine ethylester in
peptidyl-dehydrodopaquinone.
[0121] Two additional catecholoxidases may be used in these
studies: one will be extracted from the fresh byssal threads of the
mussel (Geukensia demissa) in the following manner: Byssal threads
(1-23g) are macerated then triturated by hand in a ground glass
tissue grinder with two volumes of 1M NaCl, 0.05 M Tris pH 7.5, and
0.001% Triton X-100 (Waite 1985). Insolubles are removed by
centrifugation (5000.times.G, 30 min), and the material
precipitating between 10-30% (w/v) ammonium sulfate is harvested by
centrifugation and redissolved in a small volume of high saline
buffer. Chromatography is performed consecutively on Fractogel 55S
(elution buffer=1M NaCl, 0.05 M Tris pH 7.5), Sephadex LH-60
(elution buffer=40% aqueous methanol) and Sephadex G-75 (elution
buffer 0.05 M Tris and 4M urea). Enzyme is homogeneous by SDS Page
and has a specific activity of 2040 units (Rzepecki and Waite
1989). Alternatively, the second enzyme may come from the
vitellaria of Fasciola hepatica.
Example 19
Radiolabeling of Shell Proteins to Identify New Amino Acid
Derivatives Resulting from Natural Crosslinking
[0122] F. hepatica may be labeled with radioactive isotopes
corresponding to putative nucleophiles in the shell crosslinking
process (cysteine, histidine and lysine) as well as with labeled
tyrosine and the shell protein precursors purified. Each of the
proteins will be hydrolyzed under reducing conditions as previously
described for standard amino acid analysis of DOPA proteins (Waite
and Rice-Ficht 1987) or reduced to amino acids enzymatically (Rice
and Green 1977). New peaks on the HPLC profile will be compared
with standards eluting at those positions in order to identify new
species produced by crosslinking.
Example 20
Use of Bifunctional Crosslinking Agents in Combination with
Monospecific Antisera to Study Protein-Protein Interactions in
Shell Assembly
[0123] Worms will be labeled with radioisotopes to facilitate
analysis of small quantities of protein in the following studies.
The label of choice will be determined by the protein which is to
be analyzed. VpB is void of cysteine residues although methionine
would be suitable for labeling; vpC is deficient in both sulfur
containing amino acids and would be suitably labeled with glycine
or arginine.
[0124] After labeling in vitro, worms will be isolated and
newly-formed eggs of the distal uterus will be carefully extruded.
The newly formed eggs would contain minimal native crosslinks as
yet since they remain fully positive by staining with Arnow's
reagent and yet this is the earliest stage in which one might
expect to find the vitelline proteins in their ultimate relative
positions in the shell. These immature shells may be: exposed to
reversible crosslinking reagents, shell protein precursors
extracted (that are not as yet trapped within the matrix), and the
crosslinked species identified with a monospecific antibody. The
identity may be confirmed following dissolution of the crosslink
and SDS PAGE analysis.
[0125] Any choice of cross-linking reagent must be made with
solubility in mind and the ease of introduction of the cross-linker
into the immature shell structure; to a degree this is empirical
and a number of reagents may have to be tested. An ideal
cross-linking reagent for these studies considering the composition
of the proteins to be crosslinked is the thiol cleavable
bifunctional reagent DSP [dithiobis(succinimidyl propionate)].
Since no cysteines are present in vpB or vpC and the naturally
occurring crosslinks are not labile to reducing agents, the
artificially induced crosslinks will be distinguishable from the
natural. This procedure has been applied to the study of protein
disulfide isomerase binding to immunoglobulin (Roth and Pierce
1987) through crosslinking the proteins in vivo,
immunoprecipitation of complexes with antibody directed against one
protein of the complex and analysis via SDS-PAGE (reducing gel). An
alternate approach using bis(imidoesters) has been applied to the
study of a higher order structure, the pyruvate dehydrogenase
complex of Bacillus stearothermophilus (Packman and Perham 1982).
In analysis of these associations the cleavage step (acetonitrile
and methylamine) was introduced between two dimensions of a
diagonal gel electrophoresis; proteins which migrate away from the
diagonal were previously cross-linked. Proteins may again be
identified using antibody. The goal of the project is to analyze
the proximity of the various vitelline proteins in the
three-dimensional structure and to better define the role of each
in shell architecture.
Example 21
Expression of Recombinant Eggshell Precursors in Prokaryotic
Expression Vectors
[0126] In order to obtain enough protein for study in a form which
is more soluble and manipulable, recombinant protein representing
vpA, vpB and vpC will be produced in E. coli. The E. coli host does
not carry enzymes to catalyze the oxidation of tyrosine and the
products will be unmodified. The vector of choice for production of
protein is the pGEX vector which employs the
glutathione-S-transferase gene of Schistosoma as a purification
tool (Smith and Johnson 1988). The recombinant protein will be
produced as a fusion with the GST and affinity purified on
glutathione-agarose beads. A proteolytic cleavage site has been
introduced between the GST and the foreign protein permitting
release with either factor X or thrombin. This permits a single
step purification of any of the vitelline proteins followed by the
release of the proteins from the GST carrier. The non-fused protein
can then be employed in the studies outlined below.
Example 22
Enzymatic Oxidation of Eggshell Precursors
[0127] An analysis of the purified vitelline protein precursors
reveals each of them to be post-translationally modified (tyrosine
to DOPA) to a different degree. vpC is most heavily modified
(100%); vpB is modified to a lesser extent (60%) and vpA contains
only 30% modified tyrosine. Sequence analysis has shown that with
rare exception a given tyrosine residue is modified 100% with
others completely unmodified (Waite and Rice-Ficht 1992). The basis
for this modification is uncertain since there appears to be no
absolute consensus sequence flanking the modified residues.
Recombinant protein will be enzymatically modified to varying
degrees in vitro (Waite and Rice-Ficht 1987) and specific tryptic
peptides analyzed for DOPA composition.
Example 23
Production of Polyvalent and Monoclonal Antibody for Fine Structure
Localization of Proteins Involved in Shell Production
[0128] The production of antibody directed against specific
vitelline proteins has in the past proved problematic; the problems
are based on: i) an apparent lack of antigenicity of the proteins;
and ii) antibody raised against the proteins is often directed to
the DOPA moieties producing a cross-reacting antisera. To
manufacture reagents specific for each protein we propose to
inoculate rabbits (for polyspecific sera) and mice (for monoclonal
antibody) with denatured recombinant protein representing each of
the vitelline proteins in RIBI's adjuvant. The rationale is to
raise antibody against the primary sequence of the proteins which
will be useful in distinguishing proteins in Western blot or fixed,
embedded tissue (i.e. in a denatured state). High titer sera
directed specifically against vpB and one monoclonal antibody
specific to vpB have been generated; in order to probe different
epitopes of vpB, vpC or vpA, additional reagents may be required.
Shell precursors and enzymes in the vitelline globules may be
visualized through transmission EM and immunogold tagging. The
localization of each protein to each phase of the vitelline globule
is critical to the understanding of how emulsions of the proteins
are formed and later combined prior to quinone tanning.
Example 24
Coacervation Produced Capsules Containing vpB
[0129] Capsules composed of vpB ranging from 5 to 200 microns in
size were produced through coacervation technology as follows: 200
.mu.l of phosphate buffered saline solution, pH 7.2, containing 2
mg recombinant vpB and 2 mg recombinant proline-rich protein from
F. hepatica was vortexed; 240 .mu.l of 2-isopropanol (coacervating
agent) was added dropwise while vortexing. As mixing continued, 60
.mu.l of sodium sulfate solution (30% by weight sodium sulfate in
dH2O) was added dropwise. Fixation was achieved by addition of 54
.mu.l of formaldehyde solution (37% in dH.sub.2O) while mixing for
10 minutes.
Example 25
Protein Microspheres Produced Through Oil in Water Emulsion
Technology
[0130] Encapsulation and release of small molecules, entrapment of
tritiated glycine. Protein microspheres were produced using the
following formulation: Microspheres were produced as a composite
between bovine serum albumin (Fraction V, Sigma Chemicals) and
recombinant vpB. 100ml of olive oil is stirred for 30 minutes in a
400 ml beaker using a Caframo ultra high torque stirrer at a speed
at 1200 rpm (300-1800 rpm) specific for the size of capsule to be
produced. Then 2 ml of a 126 mg/ml protein solution containing
bovine serum albumin (125 mg) and vpB (1 mg) and a compound to be
dispersed (1.25 mg glycine; 30 microcuries [.sup.3H] glycine) was
added to the oil and the stirring continued for 30 minutes at 1200
rpm. Aqueous droplets of protein in the emulsion were crosslinked
through the addition of 50 microliters of 37% formaldehyde (or
glutaraldehyde 0.1-25%) followed by stirring for an additional
15-30 minutes. Glycine, a capping agent for aldehyde groups, was
then added (125 mg/ml, 50 microliters of a 125 mg/ml solution in
phosphate buffered saline) and the emulsion stirred for an
additional 15 minutes. Microspheres were then collected through
centrifugation (10 minutes, 2400 rpm, Beckman TH-4 centrifuge) and
the microspheres washed in ether (twice in 20 ml diethyl ether) to
remove residual oil. Size is determined by oil viscosity, stirring
speed and surface properties of the encapsulant.
[0131] Release of [.sup.3H] glycine was determined by rocking the
microcarriers in a solution of phosphate buffered saline (PBS) or
in PBS:bovine serum (50:1). Samples were taken throughout a 72 hour
period and analyzed through liquid scintillation (P. Baukudumbi, K.
H. Carson, A. C. Rice-Ficht and M. J. Andrews, On the diameter and
size distribution of bovine serum albumin (BSA) microspheres,
Journal of Microencapsulation (2004)). As shown in FIG. 2, it was
found that using the present invention the release of a small
molecule, [.sup.3H] glycine, was delayed for up to eight hours with
72-75% released by that time point. The remaining 25% was released
over 72 hours.
Example 26
VpB/Albumin Protein Microsphere Utility as a Vaccine Delivery
Vehicle for Extended or Controlled Release of Tetanus Toxoid
Fragment
[0132] Microcarriers produced through the same emulsion formulation
as described for glycine entrapment were utilized in the following
studies. Groups of six mice were treated via subcutaneous injection
of encapsulated tetanus toxoid (TT; 10 micrograms per dose)
manufactured with oil in water emulsion technology (above).
Composite capsules contained the following: 74% albumin: 26% vpB. A
second group of capsules were bovine serum albumin loaded with TT
(99.99% bovine serum albumin; 10 micrograms TT) and composite
(vpB/albumin) loaded with TF (10 micrograms/dose).
[0133] The vpB microencapsulating additive was also used to entrap
tetanus toxoid for the above studies. Capsules were introduced
subcutaneously as a depot and serum antibody titers monitored for a
period of weeks using ELISA (FIG. 3). Use of 26% vpB in the
composite capsule formulation resulted in an extended period of
elevated serum antibody response to tetanus toxoid, At week 11 a
level more than 10 fold that of TT loaded albumin capsules was
observed.
Example 27
VpB/Albumin Protein Microsphere Utility as a Vaccine Delivery
Vehicle for Botulinum Toxin Fragment. Microcarriers Produced
Through the Same Emulsion Formulation as Described for Glycine
Entrapment
[0134] FIG. 4 is a graph that shows the results of botulinum toxin
encapsulated using the present invention and delivered as a depot.
Serum antibody titers for mice determined over a period of 17 weeks
are shown. Groups of 6 mice were analyzed. Results are from pooled
sera.
[0135] The hallmark of the composite capsule is the slow erosion
and the extended release profile (group 4C) when delivered as a
subcutaneous depot. Botulinum neurotoxin A (recombinant fragment C)
was delivered as a subcutaneous depot in: free form (5C, 2
micrograms per dose), and encapsulated in protein (1C-4C). 4C is a
10 micron composite capsule with 3% vpB; 97% bovine serum albumin;
2 micrograms Bot toxin). Serum antibody response with this capsule
formulation is retained at 17 weeks. This is superior to the
delivery of the toxin in a free form which directs a high serum
antibody response at 5 weeks that diminishes sharply at 7
weeks.
Example 28
Dose Dependence of the Serum Antibody Response to Encapsulated
Botulinum Toxin Fragment C
[0136] FIG. 5 is a graph that shows the immune response to doses of
botulinum neurotoxin A, fragment C trapped in the composite capsule
described above, were dose dependent. The batches of capsules are
identified by dose referring to the mg dose of capsule. The capsule
doses in milligrams contain the following doses of bot tox: 0.2
mg-0.2 micrograms of toxin; 0.6 mg-0.6 micrograms of toxin; 2.0
mg-2.0 micrograms of toxin. Individual animals received 2
micrograms per dose. Results indicate that vpB albumin composite
capsules carrying a 2.0 mg dose of capsule induce serum antibody
titers at substantially extended times when compared to lower doses
of botulinum neurotoxin A, fragment C.
Example 29
Emulsion Capsules Used to Entrap Tetanous Toxoid and Deliver
Vaccine Through a Combination of Subcutaneous/Oral Routes to
Mice
[0137] FIG. 6 is a graph that shows the antibody response of six
mice injected subcutaneously with encapsulated TT composite
capsules. Capsules were produced through emulsion technology
described above entrapping tetanus toxoid (TT, 100 micrograms per
dose). Groups of six mice were injected subcutaneously with
encapsulated TT composite capsules (tetanous toxoid 10 micrograms
per milligram of formulation/bovine serum albumin 95%/recombinant
vpB5%, G1), albumin/TT (bovine serum albumin 99.99%, tetanus toxoid
10 micrograms per milligram of formulation G2), empty composite
capsule (bovine serum albumin 95%/recombinant vpB5%, G3), tetanus
toxoid unencapsulated, 100 micrograms (G4) and saline (G5). Dosing
was performed as follows: Groups 1-3 received: 10 mg of specified
capsule formulation (containing TT 100 micrograms) delivered
subcutaneously at time 0. Mice were boosted orally at weeks 3 and 6
with 10 mg of specified formulation in 250 microliters of corn oil
and buffer (1:1). Group 4 received 100 micrograms of unencapsulated
TT at time 0 and was boosted at 3 and 6 weeks orally with 100
micrograms TT in 250 microliters of corn oil: buffer (1: 1). Group
5 received no treatment Sera were pooled for analysis and serum
antibody measured through ELISA using tetanus toxoid as antigen.
Secondary antibody detected IgG. Results indicate that the
composite formulation which contains vpB at a level of 5% and
tetanus toxoid as antigen results in an extended presentation of
antigen to the immune system and a higher serum antibody level (3
fold) at extended times when compared with a control empty
composite capsule (G3). Parallel studies with doses of 200
micrograms of tetanus toxoid revealed similar results.
[0138] FIG. 7 is a graph that shows cellular immune response as
measured by tritium uptake for the same groups as FIG. 6 at 21
weeks. Splenocyte blastogenesis from the same mice at the
conclusion of the studies indicated that a strong cell mediated
response had also been obtained when utilizing vpB as an additive
(group 1).
Example 30
Assay for the Adhesive Properties of Vitelline B Protein
[0139] Vitelline B protein (4 mg) can be dissolved in 150 ml of
Phosphate Buffered Saline (PBS-10 mM phosphate, 120 mM NaCl, pH
7.4) or 150 ml of 5 M Guanidinium HCl. This solution can be
combined with an equal volume of 30% hydrogen peroxide (25 ml of
each). The solution can be placed between the surfaces to be
adhered to one another, and placed in a 45.degree. C. oven
overnight in order to cure.
[0140] Other chemical oxidants can also be used including
chemicals, enzymes, such as mushroom tyrosinase, and buffers with
extreme pH to convert tyrosine in the vpB recombinant protein to
DOPA and DOPA quinone and impart sticky properties to the protein.
The use of a denaturant is optional, and others such as urea can be
used in place of guanidinium hydrochloride. General methods for
making proteins adhesives are taught Hwang, D. S., Yoo, H. J.,
Moon, W. K., and Cha, H. J. (2004) Applied and Environ. Micro. 70,
3352-3359, relevant portions incorporated herein by reference.
Example 31
Alginate-vpB Composite Capsules for Vaccination with Live Brucella
abortus
[0141] Capsule design 1: Bacteria
1.times.10.sup.4-1.times.10.sup.10 were suspended in 2 ml phosphate
mixed with 10 ml of a 1.5% alginate solution in a 60 ml syringe.
The syringe was attached to the inflow port of an Encapsulator
device (Inotech, Inc.) and delivered dropwise through a nozzle of
defined aperture (50-700 microns) into a CaCl.sub.2 bath (10 mM
CaCl.sub.2, 225 ml). Beads thus formed were incubated with stirring
for 5 minutes. CaCl.sub.2 was withdrawn and a solution of either
0.05% poly-L-lysine or 0.05% poly-L-lysine in CaCl.sub.2 was added
along with 0.0625-1 mg of vpB protein. This solution was stirred
for 10 minutes and withdrawn. The capsules were then washed once
for 1 minute with 100 ml MOPS buffer, then once for 5 minutes with
150 ml MOPS buffer. The MOPS buffer was withdrawn and 100 ml of a
0.03% solution of alginate was added to the beads and stirred for 5
minutes. Alginate was again withdrawn and the capsules washed with
100 ml MOPS buffer for 1 minute, drained and washed with an
additional 150 ml MOPS buffer for 5 minutes. Capsules were
collected and stored in MOPS buffer at 4.degree. C.
[0142] Capsule design 2: Alternatively, The vpB protein, at the
same concentrations indicated in capsule 1, was included with the
alginate and bacteria along with the contents of the syringe and
dropped into the CaCl.sub.2 solution. In this embodiment the vpB
was not included in the `shell`, but as component of the core. Both
applications were tested in mice.
[0143] FIG. 8 is a graph that shows the results obtained from red
deer vaccination studies conducted with capsule design 1 only using
the following approach: Groups of 4 red deer were vaccinated orally
or subcutaneously with alginate or alginate composite capsules
containing entrapped live Brucella abortus vaccine. Animals were
bled prior to commencement of the study for baseline serum titers.
Animals were vaccinated on day 1 and boosted with the same dose and
formulation at 4 weeks. Animals were challenged with the vaccine
strain on week 7 and sacrificed at week 14. The vaccination groups
are as follows: empty composite capsules (subcutaneous),
unencapsulated vaccine strain S19 (injected), alginate encapsulated
vaccine strain (injected), composite encapsulated vaccine strain
(injected), encapsulated vaccine strain (oral), composite
encapsulated vaccine strain (oral). Each animal received a dose of
7 .times.10.sup.9 -1.times.10.sup.10 organisms per dose. The
results of this study are indicated below:
[0144] Serum IgG titers of red deer at week 11 indicate that
encapsulated forms of delivery out perform the unencapsulated
vaccine with respect to maintenance of serum antibody titer. At 11
weeks the titer of animals vaccinated with composite capsules was
lower than that of alginate alone while the orally dosed animals
showed higher titers with composite capsules that those
encapsulated with alginate alone.
[0145] FIG. 9 is a graph that shows the kinetics of the induction
of humoral immunity as a result of subcutaneous vaccination.
Although the titers of animals receiving unencapsulated S19 are
falling at 7 weeks, that of both encapsulated forms is rising. The
alginate encapsulated vaccine is producing a steep increase in
titer at 4 and 7 weeks. The composite alginate/vpB encapsulated
vaccine is rising at a slower rate providing a lower level, longer
term release of the vaccine. Therefore, longer term release was
made possible by the inclusion of the vpB protein additive of the
present invention, possibly due to its slow decomposition enhancing
the performance of capsules in extended release.
Example 32
Alginate-vpB Composite Capsules for Vaccination with Live Brucella
melitensis in Mice
[0146] To determine the utility of vpB in composite capsules the
present inventors used capsule design 1 and 2 in the delivery of an
attenuated Brucella melitensis strain to mice. All animals except
group 1 received an innoculum of 1.times.10.sup.5 organisms through
intraperitoneal injection at the initiation of the study. Serum
antibody titers have been monitored weekly over a three week period
following vaccination. The groups as summarized in Table 2. The
results are shown in FIG. 10. Improved antibody titers against the
live vaccine strain are seen with the use of capsule formulation
#2, in which vpB is included with the alginate core and with the
vaccine strain. It is predicted that vpB containing formulation
will experience extended serum antibody titers and enhanced
protection over other methods. Alginate capsules, which do not
contain any vpB additive, failed to produce antibody titers as
elevated as those of vpB containing alginate capsules.
2TABLE 2 Intraperitoneal Injection of Brucella melitensis in mice
Number of Organisms animals per INOCULUM Group # Dose per dose
group MOPS buffer 1 0.1 ml 10.sup.5 10 Alginate 2 0.1 ml 10.sup.5
10 Alginate/Brucella 3 0.1 ml 10.sup.5 10 mellitensis(2*)
Alginate/vpB 4 0.1 ml 10.sup.5 10 capsule 1/ Brucella
melitensis(1*) Alginate/vpB 5 0.1 ml 10.sup.5 10 capsule 2/
Brucella melitensis(3*) Brucella 6 0.1 ml 10.sup.5 10 melitensis
unencapsulated
Example 33
Alginate-vpB Capsules for Controlled Release of an Active Protein,
Interferon tau
[0147] Capsule design 1 was used as follows. Interferon tau (from
Dr. Fuller Baser, Dept. Animal Science, TAMU) 1 ml (10.sup.8 units)
was suspended in 1 ml MOPS buffer and mixed with 5 ml of a 1.5%
alginate solution in a 60 ml syringe. The syringe was attached to
the inflow port of an Encapsulator device (Inotech, Inc.) and
delivered dropwise through a nozzle of defined aperture (50-700
microns) into a 10 mM CaCl.sub.2 bath (225 ml). Beads thus formed
were incubated with stirring for 5 minutes. CaCl.sub.2 was
withdrawn and a solution of either 0.05% poly-L-lysine or 0.05%
poly-L-lysine in CaCl.sub.2 was added in combination with 0.0625-1
mg of vpB protein. This solution was stirred for 10 minutes and
withdrawn. The capsules were then washed once for 1 minute with 100
ml MOPS buffer, then once for 5 minutes with 150 ml MOPS buffer.
The MOPS buffer was withdrawn and 100 ml of a 0.03% solution of
alginate was added to the beads and stirred for 5 minutes. Alginate
was again withdrawn and the capsules washed with 100 ml MOPS buffer
for 1 minute, drained and washed with an additional 150 ml MOPS
buffer for 5 minutes. Capsules were collected and stored in MOPS
buffer at 4.degree. C.
[0148] Encapsulated interferon tau was delivered to mice as a
treatment regimen for virally induced neurodegenerative disease.
Capsules of 400 microns in diameter containing a dose of
2.times.10.sup.6 units were delivered through intraperitoneal
injection.
[0149] SJL mice were infected with 5.times.10.sup.4 pfu BeAn strain
of Theiler's virus or mock infected with PBS. The mice were
assigned to one of the following groups (10 mice per group)
described in Table 3 below. The dose IFN will be 105 units i.p.
which has been shown to be effective in the treatment of TVID as
summarized in Table 3.
3TABLE 3 Results from Study Groups Route of Timing of Group
Infection Treatment Treatment Treatment A + IFN-.tau. i.p Daily B +
IFN-.tau. oral Daily C + Saline i.p Daily D + IFN-.tau. i.p. Twice
weekly E + IFN-.tau. oral Twice weekly F + Saline i.p. Twice weekly
G + Saline oral Twice weekly H - IFN-.tau. i.p. Daily I - IFN-.tau.
oral Daily J + Encapsulated i.p. Once biweekly IFNt
[0150] The mice have been weighed and evaluated for clinical signs
of disease at weekly intervals. Mice treated with encapsulated INFt
shown clinical inprovement comparable to that of mice receiving
daily intraperitoneal doses. The delivery of a dose of encapsulated
INFt serves to reduce the treatment regimen from once daily to once
every 2 weeks.
[0151] Controlled release of a model protein, green fluorescent
protein, from capsules of type one design have been carried out by
loading of capsules during manufacture and monitor of release
kinetics over a two week period. Results indicate release of only
5-6% of the protein within twenty four (24) hours and sustained
release of the protein over a 10 day period from capsules of type
one design. FIG. 11 shows the fluorescence profile of the gfp
released over a one week period as determined through SDS
polyacrylamide gel electrophoresis and fluorescence analysis. Lane
1 illustrates gfp released from an alginate capsule type 1 in 3
hours, lane 2, 24 hours and lane 3, 7 days. Release in a 24 hour
period is approximately 5-6% of the total encapsulated protein. The
gfp protein is the approximate molecular weight of interferon tau
and illustrates the predicted release profile of a similar protein
from this type of capsule.
[0152] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims. All publications and patent
applications mentioned in the specification are indicative of the
level of skill of those skilled in the art to which this invention
pertains. All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
[0153] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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