NASA Technical Reports Server (NTRS) 20170001265: Use of Vitelline Protein B as a Microencapsulating Additive

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by  NASA Technical Reports Server (NTRS)

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(12) United States Patent (io) Patent No.: US 9,555,073 B2
Ficht et al. (45) Date of Patent: Jan. 31, 2017
(54) USE OF VITELLINE PROTEIN B AS A OTHER PUBLICATIONS
MICROENCAPSULATING ADDITIVE
Ain et al. (2003) Alginate-based oral drug delivery system for
(75) Inventors: Allison R. Ficht, College Station, TX tuberculosis: pharmacokinetics and therapeutic effects, J.
(US); Ken Carson, Bryan, TX (US); Antimicrob. Chemother., vol. 51, pp. 931-938.*
Hitchens et al. (Dec. 2002) Annual Rept, "Orally Administered
Cynthia Sheffield, College Station, TX
Bioadherent Sustained Release Microencapsulated Vaccines", pp.
(US); John Herbert Waite, Santa 1-31.*
Barbara, CA (US) Migneault et al. (2004) Glutaraldehyde: behavior in aqueous solu-
tion, reaction with proteins, and application to enzyme crosslinking,
(73) Assignees: THE TEXAS A&M UNIVERSITY Biotechniques, vol. 37, Noi.5, pp. 790-802.*
SYSTEM, College Station, TX (US); Liu et al. (2006) Chemistry of periodate-mediated cross-linking of
3,4-dihydroxylphenylalanine-containing molecules to proteins, J.
THE REGENTS OF THE
Am. Chem. Soc., vol. 128, No. 47, pp. 15228-15235.*
UNIVERSITY OF CALIFORNIA,
Rice-Fitch et al. (1992) Eggshell precursor proteins of Fasciola
Oakland, CA (US) hepatica, L Structure and expression of vitelline protein B, Mol.
Biochem. Parasitol., vol. 54, No. 2, pp. 129-141.*
(*) Notice: Subject to any disclaimer, the term of this Tang et al. (2005) Molecular Cloning and Characterization of
patent is extended or adjusted under 35 Vitelline Precursor Protein B1 From Clonorchis Sinensis, J.
Parasitol., vol. 91, No. 6, pp. 1374-1378.*
U.S.C. 154(b) by 0 days.
Arenas-Gamboa, Angela M., et al., "Immunization with a Single
dose of a Microencapsuiateci Brucella melitensis Mutant Enhances
(21) Appl. No.: 13/407,427
Protection against Wild-Type Challenge," Infection and Immunity
Jun. 2008, p. 2448-2455.
(22) Filed: Feb. 28, 2012 Ficht, Allison, et al., "Progress Report," Jun. 30, 1990.
"File List," Published by: American Society for Microbiology,
(65) Prior Publication Data (2009).
Giletto, Anthony, `Biodegradable Bioadherent Microcapsules for
US 2012/0156287 Al Jun. 21, 2012 Orally Administered Sustained Release Vaccines," May 1997,
Annual-Phase II (Apr. 29, 1996-Apr. 28, 1997), Report AD No.
ADB226038.
Related U.S. Application Data Hwang, D. S., et al, "Expression of Functional Recombinant Mussel
Adhesive Protein Mgfp-5 in Escherichia coh," Applied and Environ
(62) Division of application No. 11/015,734, filed on Dec. Micro (2004), 70:3352-3359.
17, 2004, now abandoned. Morozova, E.V., et al., "RAPE) Variation in Two Tremode Species
(Fascila hepatica and Dicrocoelium dendriticum) from a Single
Cattle Population," Russian Journal of Genetics, vol. 88, No. 8,
(60) Provisional application No. 60/530,721, filed on Dec. 2002, pp. 977-983.
18, 2003. Rebelatto, M.C., et al., "Induction of systemic and mucosal immune
response in cattle by intranasal administration of pig serum albumin
(51) Int. Cl. in alginate mioropartioles," Veterinary Immunology and
C07K 16/02 (2006.01) immunopathology 83 (2001) 93-105.
Arnow, L. Earle, "Colorimetric Determination of the Components
C12N 15/00 (2006.01)
of 3,4- Dihydroxyphenylalanine-Tyro sine Mixtures" Laboratory of
A61K 38/17 (2006.01)
Physiological Chemistry, University of Minnesoty, Minnepolis, Jan.
A61K 9/16 (2006.01)
14, 1937.
A61K 9/50 (2006.01) Bahukudumbi, P., et al, "On the Diameter and Size Distributions of
A61K 39/00 (2006.01) Bovine Serum Albumin (BSA)-based Microspheres," J. Microen-
A61K 38100 (2006.01) capsulation, Nov. 2004, vol. 21, No. 7, 787-803.
(52) U.S. Cl. Bordier, Clement, "Phrase Separation of Integral Membrane Pro-
CPC ............. A61K 38/17 (2013.01); A61K 9/1652 teins in Triton X-114 Solution," The Journal of Biological Chem-
istry, Feb. 25, 1981, vol. 256, No. 4, pp. 1604-1607.
(2013.01); A61K 9/1658 (2013.01); A61K
Duckworth, Harry W., et al, "Physicochemical and Kinetic Proper-
9/5052 (2013.01); A61K 39/0003 (2013.01)
ties of Mushroom Tyrosinase," The Journal of Biological Chemis-
(58) Field of Classification Search try, Apr. 10, 1970, vol. 245, No. 7, pp. 1613-1625.
None
(Continued)
See application file for complete search history.
Primary Examiner Anand Desai
(56) ReferencesCited
Assistant Examiner Samuel Liu
U.S.PATENTDOCUMENTS (74) Attorney, Agent, or Firm Edwin S. Flores; Daniel
J. Chalker; Chalker Flores, LLP
4,340,588 A * 7/1982 Woodard ................... 424/252.1
5,700,909 A * 12/1997 O'Brien ................. C07K 14/47 (57) ABSTRACT
530/326
5,879,712 A * 3/1999 Bomberger et al...........424/489 The present invention includes compositions and methods
6,746,635 B2 6/2004 Mathiowitz et al. for the use of an encapsulation additive having between
2002/0053544 Al* 5/2002 Huang et al. ................. 210/650 about 0.1 to about 30 percent isolated and purified vitelline
2003/0087954 Al* 5/2003 Palepu et al. ................. 514/449
2004/0028647 Al* 2/2004 Zagury et al. ............... 424/85.1 ppr oteinn B to provide for mixed and extended release formu-
2005/0260258 Al 11/2005 Ficht et al. lations.
2006/0018918 Al * 1/2006 Chang ........................ 424/185.1
2010/0184950 Al* 7/2010 Lee et al. ...................... 530/324 11 Claims, 4 Drawing Sheets
US 9,555,073 B2
Page 2
(56) References Cited Molecular and Biochemical Parasitology, 1992, 54, 129-142,
Elsevier Science Publishers B.V.
Waite, J. H., "Presclero Eggshell Protein from the Liver Fluke
OTHER PUBLICATIONS Fasciola hepatica," Biochemistry, 1987, 26, 7819-7825.
Gyllensten, Ulf B., et at, "Generation of Single-Stranded DNA by Waite, J. H., "A Histidine-Rich Protein from the Vitellaria of the
the Polymerase Chain Reaction and its Application to Direct
Liver Fluke Fasciola hepatica," Biochemistry, 1989, 28, 6104-
Sequencing of the HLA-DQA Locus," Proc. Natl. Acad. Sci., Oct.
6110.
1988, Vo. 85, pp. 7652-7656.
Qurrat-Ul-Ain, et at, "Alginate-based Orak Drug Delivery System Waite, J. H., "Eggshell Precursor Proteins of Fasciola hepatica. II.
of Tuberculosis: Pharmacokinetics and Therapeutic Effects," Jour- Microheterogeneity in Vitelline Protein B." Molecular and Bio-
nal of Antimicrobial Chemotherapy, 2003, 51, 931-938. chemical Parasitology, 1992, 54, 143-152.
Rice-Ficht, Allison C., et at, "Eggshell Precursor of Fasciola
Hepatica, L Structure and Expression of Vitelline Protein B," * cited by examiner
U.S. Patent Jan. 31, 2017 Sheet 1 of 4 US 9,555,073 B2
VpB1
N C
FIG. 1
VpB2
C
IN
TOTAL CPMs 3H-GLY IN
BSA/VPB MICROCARRIERS
750000
700000
650000
_ II
600000 - PI
I
~ I
I
550000
COUNTS
PER MINUTE 500000
450000 -o- - PBS
400000 —*— PBS/SERUM
350000
FIG. 2
300000
0 4 S 12 16 20 24
TIME( HOURS)
ANTIBODY RESPONSE TO DEPOT DELIVERY
1.4
1.2
1.0
0.8
ANTIBODY
TITER/OD 400 0.6
0.4
0.2
0.0 FIG. 3
WEEK 1 WEEK 3 WEEK 5 WEEK 11
U.S. Patent Jan. 31, 2017 Sheet 2 of 4 US 9,555,073 B2
FIG. 4
1.0
0.9 0
GROUP 1 C
0.8 Ej
GROUP 2C
0.7
® GROUP 3C
0.6
ABSORBANCE ® GROUP 4C
0.5
OF 1:4 DILUTION
® GROUP 5C
0.4
0.3
0.2
0.1
0.0
1 2 3 4 5 7 9 13 17
TIME( WEEKS)
FIG. 5
1.0
0.9
0.2 MG DOSE
0.8
0
H MG DOSE
0.7
Ej
20 MG DOSE
0.6
ABSORBANCE
OF 1:4 DILUTION 0.1
0.4
0.3
0.2
0.1
0.0
2 3 4 5 7 9 13 17
TIME( WEEKS)
U.S. Patent Jan. 31, 2017 Sheet 3 of 4 US 9,555,073 B2
SERUM ANTIBODY LEVELS WEEK 19
1.4
1.2
1.0
06 FIG. 6
ABSORBANCE
0.4
0.2
0.0
kzsmvvr
1 2 3 4 5
ANIMAL GROUP
WEEK 21 TET 10 TRITIUM COUNTS
40000
35000
30000
25000
FIG. 7
TRITIUM COUNTS 20000
15000
10000
5000
0
1 2 3 4 5 6
ANIMAL GROUP
DIFFERENCE IN AB TITER BY ELISA
0.08
0.07
0.06 SERIES 1
0:05
OD 450 0.04
0.03
0.02
0.01
0.00
EMPTY 1NJ, INJ, ORAL ORAL
INJ S19
CAPSULES ALG S19 VPB S19 ALG S19 VPB S19
VACCINATION GROUP
FIG. 8
U.S. Patent Jan. 31, 2017 Sheet 4 of 4 US 9,555,073 B2
FIG. 9
Ab TITER THROUGH 7 WEEKS
0.090
0.080 --~.... INJ, S19
0.070 ~. INJ. AIg S19 /
0.060 --+-- INJ. VPB S19 /
0.050
0 OD 450
0.040
0.030 .~
0.020
0.010
0.000
FIG. 11 PRE-VAC 4 WEEKS 7 WEEKS
TIME
FIG. 10
ANTIBODYRESPONSE IN BALB/CMICE 1-2
WEEKSPOST INOCULATION WITHB.MELITTENSIS
0.7
0:6 ~ 1 WEEK
0.5
OD VALUE 0.4 2 WEEKS
AT 450 0.3
0.2 i
0,1
0.0
Lu
00-w ~wv~——ni ~wc~~ra c ~
''- zw z~w z w 0wQ
JZD
m. .. 1 4J JuJ J J m
m
m ¢ ,0¢.m U
W
2
INOCULUM
US 9,555,073 B2
2
USE OF VITELLINE PROTEIN B AS A glucagon. Microcapsules have also found application in the
MICROENCAPSULATING ADDITIVE food, cleaning, adhesives, fertilizers and aerospace indus-
tries to name a few.
CROSS-REFERENCE TO RELATED Albumin has been used in the preparation of microcap-
APPLICATIONS 5 sules. Two technologies have been used—coacervation, or
oil in water emulsions. These techniques have been the
This application is a divisional of U.S. Ser. No. 11/015, source of many publications, including Zimmer et al. (7.
734, filed on Dec. 17, 2004, entitled "Use of Vitelline Controlled Release 33: 31-46 (1995)); Cremers et al. (Bio-
Protein B as a Microencapsulating Additive", by inventors materials 15(1): 38-48 (1994)); Deasy (Microencapsulation
Ficht, et al., currently pending, which claims benefit under 10 and Related Drug Processes; Marcel Dekker, Inc., NY
35 U.S.C. §119(e) of provisional application U.S. Ser. No. (1984)); and Tomlinson, E. and Burger, J. J. Methods Enzy-
60/530,721, filed Dec. 18, 2003. The contents of which are mol. 112: 27-43 (1985)). Despite the efforts made towards
incorporated by reference herein in their entirety. developing microencapsulation methods, there still exists a
15 need for novel and effective materials for the encapsulation
STATEMENT REGARDING FEDERALLY of various materials.
SPONSORED RESEARCH
SUMMARY OF THE INVENTION
The government may own rights in the present invention
pursuant to grant number NCC-1-02038 from the National The compositions and methods of the present invention
20
Aeronautics and Space Administration (NASA); contract include, generally, an encapsulation additive comprising
#0300125 from the US Geological Survey, Department of between about 0.1 to about 30 percent isolated and purified
the Interior, National Park Service; and contract#DAMD17- vitelline protein B. The present inventors recognized that a
95-C-5048 from the U.S. Army Medical Research and key aspect of trematode infection and survival is the avoid-
Materiel Command. 25 ance of host immune responses. Studies as to the causes for
evasion of host immune surveillance led to the isolation and
FIELD OF THE INVENTION characterization of proteins from trematode eggshells. Upon
isolation of the genes for the trematode proteins, vitelline
The invention relates to compositions and methods for protein A (vpA), vitelline protein B (vpB) and vitelline
extended release encapsulation using additives, and more 30 protein C( vpQ and isoforms oft he same,i t was discovered,
particularly, to vitelline proteins as microencapsulation addi- as disclosed herein that the vpB protein, in particular, is
tives and uses thereof. stable at a wide range of pHs, is protease resistant and
non-immunogenic. As such, the present investigators used
INCORPORATION-BY-REFERENCE OF the vpB protein as an additive for the encapsulation and
MATERIALS FILED ON COMPACT DISC 35 extended release of, small molecules, antigens and the like.
More particularly, the encapsulation additive of the pres-
The present application includes a Sequence Listing ent invention may include the vitelline protein B from a
Trematode sp. The additive is generally non-antigenic, is
which has been submitted in ASCII format via EFS-Web and
resistant to acid and base pH or both and may allow for the
is hereby incorporated by reference in its entirety. Said
ASCII copy, created on Jul. 26, 2016, is named 40 slow release of an active agent to between about 1 hour to
about to about 8 hours, between about 1 hour to about 2
TAMU1006_seq_listing.txt and is 5K bytes in size.
weeks, and between about 1 hour to about 6 months.
Examples of the one or more active agents may include a
BACKGROUND OF THE INVENTION
pharmaceutical agent, an enzyme, a cytokine, a growth
45 promoting agent, an antibody, an antigen, a vaccine, a cell,
Without limiting the scope of the invention, its back-
a live-attenuated pathogen, a heat-killed pathogen, a virus, a
ground is described in connection with polymeric encapsu-
bacteria, a fungi, a peptide, a carbohydrate, a nucleic acid,
lation of compounds.
a lipid, mixtures and combinations thereof. The vitelline
The encapsulation of pharmaceuticals and other com- protein B may be formed into a capsule when combined by
pounds is of continuing interest, both in industry and in 50 coacervation, added into an oil-and-water emulsion, lipo-
academia. Encapsulation may be used to modify the time- somes, spray freezing, spray drying, as a component of an
release properties of a material, to alter the solubility of a alginate microcapsule or combinations thereof. Other
material and/or to provide other desirable property modifi- examples of active agents include: protein, peptide, carbo-
cations. Polymers, biopolymers and peptides have been hydrate, polysaccharide, glycoprotein, lipid, hormone,
explored for their suitability as microencapsulation agents. 55 growth factor, cytokine, interferon, receptor, antigen, aller-
Microcapsules include, generally, a core material (liquid gen, antibody, substrate, metabolite, cofactor, inhibitor,
or solid) encased in a specialized coating. Microencapsula- drug, pharmaceutical, nutrient, toxin, poison, explosive,
tion technology has found widespread use in the pharma- pesticide, chemical warfare agent, biowarfare agent, biohaz-
ceutical area, for example, coating of drugs to extend or ardous agent, infectious agent, prion, radioisotope, vitamin,
delay their release or target their release to a specific area of 6o heterocyclic aromatic compound, carcinogen, mutagen, nar-
the digestive tract. Other interesting applications include cotic, amphetamine, barbiturate, hallucinogen, waste prod-
encapsulation of ink in carbonless carbon paper, encapsu- uct, contaminant, heavy metal, virus, bacterium, Salmonella,
lation of perfumes in `scratch and snilr types of promotion- Streptococcus, Brucella, Legionella, E. coli, Guardia, Cryp-
als and encapsulation of living pancreatic cells to treat tosporidium, Rickettsia, spore, mold, yeast, algae, amoebae,
diabetes. In the case of living cells, the pore size of the 65 dinofiagellate, unicellular organism, pathogen, cell, combi-
microcapsule is selected to prevent the passage of host nations and mixtures thereof, that is encapsulated in a mixed
antibodies while allowing the free exchange of insulin and release polymer comprising between about 0.1 to about 26,
US 9,555,073 B2
3 4
about 0.1 to about 30, or even about 0.1 to about 90 weight agents, antispasmodics, antidepressant drugs, anti-diabetics,
percent isolated and purified recombinant vitelline protein anorectic drugs, anti-allergenics, decongestants, expecto-
B. rants, antipyretics, antimigrane, anti-malarials, anti-ulcer-
Yet another embodiment of the present invention may be ative, peptides, anti-estrogen, anti-hormone agents, antiulcer
a mixed release pharmaceutical formulation with one or 5 agents, anesthetic agent, drugs having an action on the
more active agents microencapsulated in a mixed release central nervous system or combinations thereof. The poly-
polymer and between about 0.1 to 26 percent isolated and mer may be a biocompatible and/or biodegradable polymer
purified vitelline protein B. A polymer selected from the selected from: poly(ethylene glycol), poly(ethylene oxide),
group consisting of cellulose, ethylcellulose, methylcellu- poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethylox-
lose, propylcellulose, methoxypropylcellulose, cellulose io azoline), poly(ethylene oxide)-co-poly(propylene oxide)
nitrate, poly(vinyl alcohol), poly(vinyl chloride), polysty- block copolymers, polysaccharides, carbohydrates, proteins
rene, polyethylene, polypropylene, poly(ethylene-co-vinyl and combinations thereof.
acetate), poly(hydroxybutyric acid), poly(hydroxyvalerianic A mixed release pharmaceutical formulation using the
acid-co-hydroxybutyric acid), poly(lactic acid), poly(gly- vpB additive of the present invention may include one or
colic acid), poly(lactic acid-co-glycolic acid), 15 more active agents selected from protein, peptide, carbohy-
poly(epsilon (-caprolactones), poly(epsilon(-caprolactone- drate, polysaccharide, glycoprotein, lipid, hormone, growth
co-DL-lactic acid), poly(maleic anhydride), polyamides, factor, cytokine, interferon, receptor, antigen, allergen, anti-
gelatin, chitosan, collagen, poly(hydroxyalkyl)-L-gluta- body, substrate, metabolite, cofactor, inhibitor, drug, phar-
mines, poly(gamma(-ethyl-L-glutaminate-co-glutamic maceutical, nutrient, toxin, poison, explosive, pesticide,
acid), poly(L-leucine-co-L-aspartic acid), poly(proline-co- 20 chemical warfare agent, biowarfare agent, biohazardous
glutamic acid), poly(alkyl 2-cyanoacrylates), polyurethanes, agent, infectious agent, prion, radioisotope, vitamin, hetero-
poly(methyl methacrylate), poly(methyl methacrylate-co- cyclic aromatic compound, carcinogen, mutagen, narcotic,
methacrylic acid) and poly(methacrylate-co-hydroxypropyl amphetamine,b arbiturate, hallucinogen, waste product, con-
methacrylate). taminant, heavy metal, virus, bacterium, Salmonella, Strep-
Other examples of active agents may include a pharma- 25 tococcus, Brucella, Legionella, E. coli, Guardia, Cryptospo-
ceutical agent, an enzyme, a cytokine, a growth promoting ridium, Rickettsia, spore, mold, yeast, algae, amoebae,
agent, an antibody, an antigen, a vaccine, a cell, a live- dinoflagellate, unicellular organism, pathogen, cell, combi-
attenuated pathogen, a heat-killed pathogen, a virus, a bac- nations and mixtures thereof, that is encapsulated in a mixed
teria, a fungi, a peptide, a carbohydrate, a nucleic acid, a release polymer and between about 0.1 to 26 weight percent
lipid, mixtures and combinations thereof. 30 isolated and purified vitelline protein B.
The present invention may also include a method of Yet another embodiment of the present invention is a
encapsulation that includes the steps of mixing one or more vaccine in which one or more antigens are encapsulated in
active agents with one or more cross-linkable monomers and a mixed release with between about 0.1 to 26 weight percent
a vitelline protein B, wherein the vitelline protein B modifies isolated and purified vitelline protein B. The antigen may be
the release profile of the one or more active agents. Alter- 35 selected from: protein, peptide, carbohydrate, polysaccha-
natively, the method of making an extended release formu- ride, glycoprotein, lipid, hormone, growth factor, cytokine,
lation may include the steps of mixing one or more active interferon, receptor, antigen, allergen, antibody, substrate,
agents with alginate and vitelline protein B, wherein the metabolite, cofactor, inhibitor, drug, pharmaceutical, nutri-
vitelline protein B modifies the release profile of one or more ent, toxin, poison, explosive, pesticide, chemical warfare
of the active agents. The one or more active agents are 4o agent, biowarfare agent, biohazardous agent, infectious
released for between about 1 hour to about 8 hours, between agent, prion, radioisotope, vitamin, heterocyclic aromatic
about 1 hour to about 2 weeks, and between about 1 hour to compound, carcinogen, mutagen, narcotic, amphetamine,
about 6 month and the vitelline protein B comprises a barbiturate, hallucinogen, waste product, contaminant,
Trematode sp. protein that is non-antigenic, is resistant to heavy metal, virus, bacterium, Salmonella, Streptococcus,
acid pH, resistant to basic pH or combinations thereof. The 45 Brucella, Legionella, E. coli, Guardia, Cryptosporidium,
method may also include the step of mixing poly-L lysine Rickettsia, spore, mold, yeast, algae, amoebae, dinoflagel-
with the vitelline protein B, wherein the ratio of the poly-L late, unicellular organism, pathogen, cell, combinations and
lysine to vitelline protein B is between about 30:70 to 70:30 mixtures thereof. Yet another embodiment of the present
weight to weight. invention is an adhesive having between about 1 to about 90
Yet another embodiment of the present invention is a 50 percent isolated and purified vitelline protein B.
mixed release pharmaceutical formulation in which one or The vpB additive and methods disclosed herein may be
more one or more pharmaceutical agents is encapsulated in used for the micro and nano-encapsulation of a number of
a mixed release polymer comprising between about 0.1 to 26 active agents, e.g., peptides, proteins, aptamers, oligonucle-
weight percent isolated and purified vitelline protein B. otides, carbohydrates, lipids, glycolipids, glycoproteins,
Examples of pharmaceutical agents include: steroids, respi- 55 anti-obesity drugs, nutraceuticals, cortico steroids, elastase
ratory agents, sympathomimetics, local anesthetics, antimi- inhibitors, analgesics, anti-fungals, antibiotics, antibodies,
crobial agents, antiviral agents, antifungal agents, antihel- antigens, oncology therapies, anti-emetics, analgesics, car-
minthic agents, insecticides, antihypertensive agents, diovascular agents, anti-inflammatory agents, antigens, anti-
antihypertensive diuretics, cardiotonics, coronary vasodila- helmintics, anti-arrhythmic agents, antibiotics, antibodies,
tors, vasoconstrictors, (3-blockers, antiarrhythmic agents, 6o anticoagulants, antidepressants, antidiabetic agents, antiepi-
calcium antagonists, anti-convulsants, agents for dizziness, leptics, antihistamines, antihypertensive agents, antimusca-
tranquilizers, antipsychotics, muscle relaxants, drugs for rinic agents, antimycobacterial agents, antineoplastic agents,
Parkinson's disease, respiratory agents, hormones, non- immunosuppressants, antithyroid agents, antiviral agents,
steroidal hormones, antihormones, vitamins, antitumor anxiolytics, sedatives, astringents, beta-adrenoceptor block-
agents, miotics, herb medicines, antimuscarinic, inter- 65 ing agents, blood products and substitutes, cardiac inotropic
fereons, immunokines, cytokines, muscarinic cholinergic agents, contrast media, cortico steroids, cough suppressants,
blocking agents, mydriatics, psychic energizers, Immoral diagnostic agents, diagnostic imaging agents, diuretics, dop-
US 9,555,073 B2
T
aminergics, haemostatics, immunological agents (cytokines, be appreciated that the present invention provides many
lymphokines), lipid regulating agents, muscle relaxants, applicable inventive concepts that can be embodied in a
parasympathomimetics, parathyroid calcitonin and biphos- wide variety of specific contexts. The specific embodiments
phonates, prostaglandins, radio-pharmaceuticals, sex hor- discussed herein are merely illustrative of specific ways to
mones,i nsecticides, anti-allergic agents, stimulants and ano- 5 make and use the invention and do not delimit the scope of
retics, sympathomimetics, thyroid agents, vasodilators, and the invention.
xanthines, and derivatives, salts and combinations thereof. To facilitate the understanding of this invention, a number
The skilled artisan will recognize that a number of agents
of terms are defined below. Terms defined herein have
may be used and/or delivered in combination with the
meanings as commonly understood by a person of ordinary
present invention, including, organic or inorganic molecules
io skill in the areas relevant to the present invention. Terms
(small or large), second messengers, nucleic acids (natural,
such as "a"," an" and "the" are not intended to refer to only
non-natural and derivatives thereof), amino acids (natural,
a singular entity, but include the general class of which a
non-natural and derivatives thereof), carbohydrates (mono-
specific example may be used for illustration. The terminol-
meric or oligomeric), lipids, cells, cell fragments, glycopro-
ogy herein is used to describe specific embodiments of the
teins, nutrients, vitamins, etc.
The vpB and related proteins oft he present invention may 15 invention, but their usage does not delimit the invention,
also be used in a method of treating an animal, the method except as outlined in the claims.
in which the mixed release formulation of the present The term "additive" as used herein is used to describe vpB
invention is administered to a mammal. In an alternative protein, variants thereof, and proteins and peptides that have
embodiment, the vpB protein may also be used as a bent core a similar protein composition to produce encapsulants that
mesogen piezoelectric component. 20 affect the release of an agent trapped in or about the additive,
e.g., when the additive is mixed with a polymeric or other
BRIEF DESCRIPTION OF THE FIGURES composition to form a micro or nanocapsule. It has been
found, as described in detail hereinbelow, that the Vitelline
For a more complete understanding of the features and protein B is a member of a family of proteins with variable
advantages of the present invention, reference is now made
25 sequences, however, the amino acid composition is fixed.
to the detailed description of the invention along with the
The Vitelline protein B may be closed and/or isolated by
accompanying figures and in which:
purification from, e.g., Fasciola hepatica. Alternatively, the
FIG. 1 is a map that summarizes the divergence of amino
Vitelline protein B may be made synthetically, by recombi-
acid sequence between vpB family members: hatched lines
nant methods and combinations thereof.
indicate areas of highly divergent amino acid sequence;
FIG. 2 is a graph that shows the release of a small 30 The term "immediate release" as used herein is used to
describe a release profile to effect delivery of an active as
molecule, [3H] glycine, encapsulated using the additive of
the present invention; soon as possible, that is, as soon as practically made avail-
FIG. 3 is a graph that shows the antibody titer of mice able to an animal, whether in active form, as a precursor
treated via subcutaneous injection of encapsulated tetanus and/or as a metabolite. Immediate release may also be
toxoid; 35 defined functionally as the release of over 80 to 90 percent
FIG. 4 is a graph that shows the antibody titer of mice (%)o f the active ingredient within about 60, 90, 100 or 120
treated via subcutaneous injection with botulinum toxin minutes or less.
encapsulated using the present invention and delivered as a The terms "extended release" and "delayed release" as
depot; used herein is used to define a release profile to effect
FIG.5 is a graph that shows the immune response to doses 4o delivery of an active over an extended period of time,
of botulinum neurotoxin A, fragment C trapped in the defined herein as being between about 60 minutes and about
composite capsule was dose dependent; 2, 4,6 or even 8 hours. Extended release may also be defined
FIG. 6 is a graph that shows the antibody response of six functionally as the release of over 80 to 90 percent( %)o f the
mice injected subcutaneously with encapsulated TT (tetanus active ingredient after about 60 minutes and about 2, 4, 6 or
toxoid) composite capsules; 45 even 8 hours. Extended release as used herein may also be
FIG. 7 is a graph that shows cellular immune response as defined as making the active ingredient available to the
measured by tritium uptake for the same groups as FIG. 6 at patient or subject regardless of uptake, as some actives may
21 weeks; never be absorbed by the animal. Various extended release
FIG.8 is a graph that shows the results obtained from Red dosage forms may be designed readily by one of skill in art
deer vaccination studies; 5o as disclosed herein to achieve delivery to both the small and
FIG. 9 is a graph that shows the kinetics of Immoral large intestines, to only the small intestine, or to only the
immunity with subcutaneous vaccination of the present large intestine, depending upon the choice of coating mate-
invention; rials and/or coating thickness.
FIG. 10 is a graph of serum antibody levels in Brucella "Extended release" and "delayed release" formulations
melitensis immunized mice; and 55 may be prepared and delivered using the Vitelline protein B
FIG. 11 shows the fluorescence profile of green fluores- or variants thereof to control the release of an agent. The
cent protein released over a one week period as determined Vitelline protein B protein may act alone or in combination
through SDS polyacrylamide gel electrophoresis and fluo- with other compounds that delay release of an active, e.g., a
rescence analysis. Lane I illustrates green fluorescent pro- coating, a capsule or mixture with controlled, delayed or
tein (gfp)r eleased from an alginate capsule type I in 3 hours, 60 extended release polymers. Any coatings should be applied
lane 2, 24 hours and lane 3, 7 days. to a sufficient thickness such that the entire coating does not
dissolve in the gastrointestinal fluids at pH below about 5,
DETAILED DESCRIPTION OF THE but does dissolve at pH about 5 and above. It is expected that
INVENTION any anionic polymer exhibiting a pH-dependent solubility
65 profile can be used as an enteric coating in the practice oft he
While the making and using of various embodiments of present invention to achieve delivery to the lower gastroin-
the present invention are discussed in detail below, it should testinal tract. Polymers and compatible mixtures thereof
US 9,555,073 B2
7 8
may be used to provide the coating for the delayed or the mal in need of such treatment. The therapeutically effective
extended release of active ingredients, and some of their amount will vary depending upon the subject and disease
properties, include, but are not limited to: shellac, also called condition being treated, the weight and age of the subject,
purified lac, a refined product obtained from the resinous the severity of the disease condition, the manner of admin-
secretion of an insect. This coating dissolves in media of 5 istration and the like, which can readily be determined by
pH>7. one of ordinary skill in the art. The amount of active agent
As used herein, the term "enveloped pharmaceutical" and the need and amount of carriers and/or excipients are
means a capsule, a suppository, a gel cap, a softgel, a disclosed, simply by way of example, by Remington's
lozenge, a sachet or even a fast dissolving wafer. As used Pharmaceutical Sciences, 19th edition, 1995, Ed. Gennaro,
herein the term "carrier" is used to describe a substance, io relevant portions incorporated herein by reference. The term
whether biodegradable or not, that is physiologically accept- "treatment" or "treating" means any treatment of a disease
able for human or animal use and may be pharmacologically in a mammal, including: (i) preventing the disease, that is,
active or inactive. causing the clinical symptoms of the disease not to develop;
The vpB additive of the present invention may be used in (ii) inhibiting the disease, that is, arresting the development
conjunction with a wide variety of dosage forms, e.g., 15 of clinical symptoms; and/or (iii) relieving the disease, that
solution, suspension, cream, ointment, lotion, capsule, is, causing the regression of clinical symptoms.
caplet, softgel, gelcap, suppository, enema, elixir, syrup, The additive of the present invention may be used in
emulsion, film, granule, gum, insert, jelly, foam, paste, conjunction with one or more carriers. As used herein the
pastille, pellet, spray, troche, lozenge, disk, magma, poul- terms "carrier" and "pharmaceutically acceptable carrier"
tice, or wafer and the like. 20 include any and all solvents, dispersion media, coatings,
The vpB additive of the present invention may be used to antibacterial and antifungal agents, isotonic and absorption
delivery active pharmaceutical agents. As used herein, delaying agents and the like. The use of such media and
"pharmaceutically" and/or "pharmacologically acceptable" agents for pharmaceutically active substances is well known
refer to molecular entities and/or compositions that do not in the art. Except insofar as any conventional media or agent
produce an adverse, allergic and/or other untoward reaction 25 is incompatible with the active ingredient, its use in the
when administered to an animal, as appropriate. One distinct therapeutic compositions is contemplated. Supplementary
advantage of vpB is that it does not generally cause an active ingredients can also be incorporated into the compo-
adverse, allergic and/or other untoward reaction when sitions.
administered to an animal. As used herein, the term "active ingredient(s)," "pharma-
As used herein, "pharmaceutically acceptable carrier" 30 ceutical ingredient(s)," "active agents" and "bioactive
may include any and/or all solvents, dispersion media, agent" are defined as drugs and/or pharmaceutically active
coatings, antibacterial and/or antifungal agents, isotonic ingredients. The present invention may be used to encapsu-
and/or absorption delaying agents and/or the like. The use of late, attach, bind or otherwise be used to affect the storage,
such media and/or agents for pharmaceutical active sub- stability, longevity and/or release of any of the following
stances is well known in the art. Except insofar as any 35 drugs as the pharmaceutically active agent in a composition.
conventional media and/or agent is incompatible with the One or more of the following bioactive agents may be
active ingredient, its use in the therapeutic compositions is combined with the vpB additive disclosed herein: Analgesic
contemplated. Supplementary active ingredients can also be anti-inflammatory agents such as, acetaminophen, aspirin,
incorporated into the compositions. For administration, salicylic acid, methyl salicylate, choline salicylate, glycol
preparations should meet sterility, pyrogenicity, general 40 salicylate, 1-menthol, camphor, mefenamic acid, fluphe-
safety and/or purity standards as required by FDA Office of namic acid, indomethacin, diclofenac, alclofenac, ibuprofen,
Biologics standards. ketoprofen, naproxene, pranoprofen, fenoprofen, sulindac,
As used herein, the term "therapeutically effective dos- fenbufen, clidanac, flurbiprofen, indoprofen, protizidic acid,
age" is used to describe the amount that reduces the amount fentiazac, tolmetin, tiaprofenic acid, bendazac, bufexamac,
of symptoms of the condition in the infected subject by at 45 piroxicam, phenylbutazone, oxyphenbutazone, clofezone,
least about 20%, more preferably by at least about 40%, pentazocine, mepirizole and the like.
even more preferably by at least about 60%, and still more Drugs having an action on the central nervous system, for
preferably by at least about 80% relative to untreated example sedatives, hypnotics, antianxiety agents, analgesics
subjects. Often, for pediatric doses the amount will be half and anesthetics, such as, chloral, buprenorphine, naloxone,
or less of the adult dose. For example, the efficacy of a 5o haloperidol, fluphenazine, pentobarbital, phenobarbital,
compound may be evaluated in an animal model system that secobarbital, amobarbital, cydobarbital, codeine, lidocaine,
may be predictive of efficacy in treating the disease in tetracaine, dyclonine, dibucaine, cocaine, procaine, mepiva-
humans. Bioactive compounds are administered at a thera- caine, bupivacaine, etidocaine, prilocalne, benzocaine, fen-
peutically effective dosage sufficient to treat a condition tanyl, nicotine and the like. Local anesthetics such as,
associated with a condition in a subject. 55 benzocaine, procaine, dibucaine, lidocaine and the like.
The terms "amount," "pharmaceutically effective Antihistaminics or antiallergic agents such as, diphenhy-
amount" and "therapeutically effective amount" as used dramine, dimenhydrinate, perphenazine, triprolidine, pyril-
herein refer to a quantity or to a concentration as appropriate amine, chlorcyclizine, promethazine, carbinoxamine,
to the context. The amount of an active agent or drug that tripelennamine, brompheniramine, hydroxyzine, cyclizine,
constitutes a pharmaceutically or therapeutically effective 60 meclizine, clorprenaline, terfenadine, chlorpheniramine and
amount varies according to factors such as the potency of the the like. Anti-allergenics such as, antazoline, methapyrilene,
particular drug, the route of administration of the formula- chlorpheniramine, pyrilamine, pheniramine and the like.
tion, and the mechanical system used to administer the Decongestants such as, phenylephrine, ephedrine, naphazo-
formulation as will be known to the skilled artisan. For line, tetrahydrozoline and the like.
example, a pharmaceutically or therapeutically effective 65 Antipyretics such as, aspirin, salicylamide, non-steroidal
amount is that dosage of active agents that when released is anti-inflammatory agents and the like. Antimigrane agents
sufficient to effect treatment, when administered to a mam- such as, dihydroergotamine, pizotyline and the like.