U.S. patent application number 13/453149 was filed with the patent office on 2012-08-16 for pharmaceutical composition of nanoparticles.
Invention is credited to Er-Yuan Chuang, Ho-Ngoc Nguyen, Kiran Sonaje, Hsing-Wen Sung, Hosheng Tu.
Application Number | 20120207845 13/453149 |
Document ID | / |
Family ID | 46148011 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120207845 |
Kind Code |
A1 |
Sung; Hsing-Wen ; et
al. |
August 16, 2012 |
PHARMACEUTICAL COMPOSITION OF NANOPARTICLES
Abstract
The invention relates to a method for treating disorders or
diseases of a tight junction comprising delivering a pharmaceutical
composition of nanoparticles to the tight junction, wherein the
nanoparticles consist of positively charged chitosan, a negatively
charged substrate, optionally a zero-charge compound, and at least
one bioactive agent for treating said disorders or diseases of the
tight junction of an animal subject.
Inventors: |
Sung; Hsing-Wen; (Hsinchu,
TW) ; Sonaje; Kiran; (Hsinchu, TW) ; Nguyen;
Ho-Ngoc; (Hsinchu, TW) ; Chuang; Er-Yuan;
(Hsinchu, TW) ; Tu; Hosheng; (Newport Beach,
CA) |
Family ID: |
46148011 |
Appl. No.: |
13/453149 |
Filed: |
April 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13373995 |
Dec 7, 2011 |
8192718 |
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13453149 |
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13068535 |
May 13, 2011 |
8084493 |
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13373995 |
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12931202 |
Jan 26, 2011 |
7993624 |
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13068535 |
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12800848 |
May 24, 2010 |
7879313 |
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12931202 |
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12321855 |
Jan 26, 2009 |
7871988 |
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12800848 |
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12286504 |
Sep 30, 2008 |
7604795 |
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12321855 |
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12151230 |
May 5, 2008 |
7541046 |
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12286504 |
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11398145 |
Apr 5, 2006 |
7381716 |
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12151230 |
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11284734 |
Nov 21, 2005 |
7282194 |
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11398145 |
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11029082 |
Jan 4, 2005 |
7265090 |
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11284734 |
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Current U.S.
Class: |
424/497 ;
514/179; 514/249; 514/254.07; 514/263.21; 514/263.3; 514/31;
514/383; 514/5.9; 514/567; 977/773; 977/795; 977/906 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 29/00 20180101; A61K 9/5146 20130101; A61K 47/645 20170801;
A61P 3/10 20180101; A61K 31/445 20130101; A61K 31/13 20130101; A61K
9/0043 20130101; A61K 31/55 20130101; A61P 25/28 20180101; A61K
9/5161 20130101; A61P 25/08 20180101; A61P 31/10 20180101; A61P
39/06 20180101 |
Class at
Publication: |
424/497 ;
514/567; 514/179; 514/263.21; 514/249; 514/263.3; 514/31;
514/254.07; 514/383; 514/5.9; 977/773; 977/795; 977/906 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/573 20060101 A61K031/573; A61K 31/52 20060101
A61K031/52; A61K 31/519 20060101 A61K031/519; A61K 31/7048 20060101
A61K031/7048; A61K 31/496 20060101 A61K031/496; A61K 31/4196
20060101 A61K031/4196; A61K 38/28 20060101 A61K038/28; A61P 25/08
20060101 A61P025/08; A61P 29/00 20060101 A61P029/00; A61P 39/06
20060101 A61P039/06; A61P 31/10 20060101 A61P031/10; A61P 25/00
20060101 A61P025/00; A61P 25/28 20060101 A61P025/28; A61P 3/10
20060101 A61P003/10; A61K 31/196 20060101 A61K031/196 |
Claims
1-20. (canceled)
21. A method of delivering at least one bioactive agent to an
animal subject by circumventing a first-pass liver metabolism, the
method comprising: (a) loading said bioactive agent in
nanoparticles; and (b) delivering said nanoparticles via a
parenteral route to said animal subject, wherein the nanoparticles
consist of positively charged chitosan, a negatively charged
substrate, optionally a zero-charge compound, and said bioactive
agent.
22. The method of claim 21, wherein said parenteral route is a
nasal pathway from the nose via olfactory mucosa to a brain tissue
via cerebrospinal fluid (CSF).
23. The method of claim 21, wherein the negatively charged
substrate is polyglutamic acid (PGA).
24. The method of claim 23, wherein said PGA is selected from the
group consisting of .gamma.-PGA, .alpha.-PGA, derivatives of PGA,
salts of PGA, and combinations thereof.
25. The method of claim 23, wherein said PGA is a PGA-complexone
conjugate.
26. The method of claim 25, wherein said complexone of the
PGA-complexone conjugate is selected from the group consisting of
DTPA (diethylene triamine pentaacetic acid), EDTA (ethylene diamine
tetra acetate), IDA (iminodiacetic acid), NTA (nitrilotriacetic
acid), EGTA (ethylene glycol tetraacetic acid), BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic, acid), DOTA
(1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid), and
NOTA (2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid).
27. The method of claim 21, wherein said nanoparticles are treated
with an enteric coating polymer.
28. The method of claim 21, wherein said zero-charge compound is a
permeation enhancer.
29. The method of claim 28, wherein said permeation enhancer is
selected from the group consisting of bile salts, surfactants,
medium-chain fatty acids, phosphate esters, and chitosan.
30. The method of claim 21, wherein said chitosan is selected from
the group consisting of N-trimethyl chitosan (TMC), low
MW-chitosan, EDTA-chitosan, pegylated chitosan (PEG-chitosan),
mono-N-carboxymethyl chitosan (MCC), chitosan derivatives, and
combinations thereof.
31. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of an anti-epileptic drug,
anti-inflammatory drug, meningitis antagonist, and
anti-oxidant.
32. The method of claim 31, wherein said anti-inflammatory drug is
selected from the group consisting of mesalazine, prednisone, a TNF
inhibitor, azathioprine (Imuran), methotrexate, 6-mercaptopurine,
nystatin, antifungal agent, itraconazole, and fluconazole.
33. The method of claim 21, wherein said bioactive agent is
selected from the group consisting of an antagonist for treatment
of multiple sclerosis, neuromyelitis optica, late-stage
neurological trypanosomiasis, progressive multifocal
leukoencephalopathy, HIV encephalitis, and Alzheimer's
diseases.
34. The method of claim 21, wherein said nanoparticles are
freeze-dried, thereby said nanoparticles being in a powder
form.
35. The method of claim 21, wherein said freeze-dried nanoparticles
are being re-constituted with sterile water prior to being
delivered to nose of said animal subject.
36. The method of claim 21, wherein said nanoparticles are
collapsed nanoparticles.
37. The method of claim 21, wherein said nanoparticles have a mean
particle size between about 50 and 500 nanometers.
38. The method of claim 21, wherein said bioactive agent is insulin
or an insulin analog.
39. The method of claim 21, wherein said bioactive agent is an
anti-diabetic drug.
40. The method of claim 21, wherein said nanoparticles are formed
via a simple and mild ionic-gelation process.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation application of U.S. patent
application Ser. No. 13/373,995 filed Dec. 7, 2011, which is a
continuation-in-part application of U.S. patent application Ser.
No. 13/068,535, filed May 13, 2011, now U.S. Pat. No. 8,084,493,
which is a continuation-in-part application of U.S. patent
application Ser. No. 12/931,202, filed Jan. 26, 2011, now U.S. Pat.
No. 7,993,624, which is a continuation application of U.S. patent
application Ser. No. 12/800,848, filed May 24, 2010, now U.S. Pat.
No. 7,879,313, which is a continuation-in-part application of U.S.
patent application Ser. No. 12/321,855, filed Jan. 26, 2009, now
U.S. Pat. No. 7,871,988, which is a continuation-in-part
application of U.S. patent application Ser. No. 12/286,504, filed
Sep. 30, 2008, now U.S. Pat. No. 7,604,795, which is a
continuation-in-part application of U.S. patent application Ser.
No. 12/151,230, filed May 5, 2008, now U.S. Pat. No. 7,541,046,
which is a continuation-in-part application of U.S. patent
application Ser. No. 11/398,145, filed Apr. 5, 2006, now U.S. Pat.
No. 7,381,716, which is a continuation-in-part application of U.S.
patent application Ser. No. 11/284,734, filed Nov. 21, 2005, now
U.S. Pat. No. 7,282,194, which is a continuation-in-part
application of U.S. patent application Ser. No. 11/029,082, filed
Jan. 4, 2005, now U.S. Pat. No. 7,265,090, the entire contents of
which are incorporated herein by reference. This application also
claims the benefits of a provisional patent application Ser. No.
61/269,424, filed Jun. 24, 2009.
FIELD OF THE INVENTION
[0002] The present invention is related to general uses of an oral
drug delivery vehicle of nanoparticles comprising chitosan, a
negatively charged substrate and a bioactive agent for treating
disorders or disease of a tight junction of an animal subject.
BACKGROUND OF THE INVENTION
[0003] Production of pharmaceutically bioactive peptides and
proteins in large quantities has become feasible (Biomacromolecules
2004; 5:1917-1925). The oral route is considered the most
convenient way of administering drugs for patients or an animal
subject. Nevertheless, the intestinal epithelium is a major barrier
to the absorption of hydrophilic drugs such as peptides and
proteins (J. Control. Release 1996; 39:131-138). This is because
hydrophilic drugs cannot easily diffuse across the cells through
the lipid-bilayer cell membranes. Attentions have been given to
improving paracellular transport of hydrophilic drugs (J. Control.
Release 1998; 51:35-46). However, the transport of hydrophilic
molecules via the paracellular pathway is, however, severely
restricted by the presence of tight junctions that are located at
the luminal aspect of adjacent epithelial cells (Annu. Rev. Nutr.
1995; 15:35-55). These tight junctions form a barrier that limits
the paracellular diffusion of hydrophilic molecules. The structure
and function of tight junctions is described, inter alia, in Ann.
Rev. Physiol. 1998; 60:121-160 and in Ballard T S et al., Annu.
Rev. Nutr. 1995; 15:35-55. Tight junctions do not form a rigid
barrier but play an important role in the diffusion through the
intestinal epithelium from lumen to bloodstream and vice versa.
[0004] Movement of solutes between cells, through the tight
junctions that bind cells together into a layer such as the
epithelial cells of the gastrointestinal tract, is termed
paracellular transport. Paracellular transport is passive.
Paracellular transport is dependent on electrochemical gradients
generated by transcellular transport and solvent drag through tight
junctions. Tight junctions form an intercellular barrier that
separates the apical and basolateral fluid compartments of a cell
layer. Movement of a solute through a tight junction from apical to
basolateral compartments depends on the permeability of the tight
junction for that solute.
[0005] Polymeric nanoparticles have been widely investigated as
carriers for drug delivery (Biomaterials 2002; 23:3193-3201). Much
attention has been given to the nanoparticles made of synthetic
biodegradable polymers such as poly-.epsilon.-caprolactone and
polylactide due to their biocompatibility (J. Drug Delivery 2000;
7:215-232; Eur. J. Pharm. Biopharm. 1995; 41:19-25). However, these
nanoparticles are not ideal carriers for hydrophilic drugs because
of their hydrophobic property. Some aspects of the invention relate
to a novel nanoparticle system, composed of hydrophilic chitosan
and poly(glutamic acid) hydrogels; the nanoparticles are prepared
by a simple ionic-gelation method. This technique is promising as
the nanoparticles are prepared under mild conditions without using
harmful solvents. It is known that organic solvents may cause
degradation of peptide or protein drugs that are unstable and
sensitive to their environments (J. Control. Release 2001;
73:279-291).
[0006] Following the oral drug delivery route, protein drugs are
readily degraded by the low pH of gastric medium in the stomach.
The absorption of protein drugs following oral administration is
challenging due to their high molecular weight, hydrophilicity, and
susceptibility to enzymatic inactivation. Protein drugs at the
intestinal epithelium cannot partition into the hydrophobic
membrane, leaving only the epithelial barrier via the paracellular
pathway. However, the tight junction forms a barrier that limits
the paracellular diffusion of hydrophilic molecules.
[0007] Chitosan (CS), a cationic polysaccharide, is generally
derived from chitin by alkaline deacetylation (J. Control. Release
2004; 96:285-300). It was reported from literature that CS is
non-toxic and soft-tissue compatible (Biomacromolecules 2004;
5:1917-1925; Biomacromolecules 2004; 5:828-833). Additionally, it
is known that CS has a special property of adhering to the mucosal
surface and transiently opening the tight junctions between
epithelial cells (Pharm. Res. 1994; 11:1358-1361). Most
commercially available CSs have a quite large molecular weight (MW)
and need to be dissolved in an acetic acid solution at a pH value
of approximately 4.0 or lower, which is somewhat impractical.
However, there are potential applications of CS in which a low MW
would be essential. Given a low MW, the polycationic characteristic
of CS can be used together with a good solubility at a pH value
close to physiological ranges (Eur. J. Pharm. Biopharm. 2004;
57:101-105). Loading of peptide or protein drugs at physiological
pH ranges would preserve their bioactivity. On this basis, a low-MW
CS, obtained by depolymerizing a commercially available CS using
cellulase, is disclosed herein to prepare nanoparticles of the
present invention.
[0008] Thanou et al. reported chitosan and its derivatives as
intestinal absorption enhancers (Adv Drug Deliv Rev 2001;
50:S91-S101). Chitosan, when protonated at an acidic pH, is able to
increase the paracellular permeability of peptide drugs across
mucosal epithelia. Co-administration of chitosan or trimethyl
chitosan chloride with peptide drugs were found to substantially
increase the bioavailability of the peptide in animals compared
with administrations without the chitosan component.
[0009] The .gamma.-PGA, an anionic peptide, is a natural compound
produced as capsular substance or as slime by members of the genus
Bacillus (Crit. Rev. Biotechnol. 2001; 21:219-232). .gamma.-PGA is
unique in that it is composed of naturally occurring L-glutamic
acid linked together through amide bonds. It is reported from
literature that this naturally occurring .gamma.-PGA is a
water-soluble, biodegradable, and non-toxic polymer. A polyamine
carboxylic acid (complexone), such as diethylene triamine
pentaacetic acid, has showed enzyme resistant property. It is
clinical beneficial to co-incorporate a PGA-complexone conjugate
and a peptide or protein drug in an oral drug delivery with reduced
enzymatic effect.
SUMMARY OF THE INVENTION
[0010] It is one object of the present invention to provide a
novel, unique nanoparticle system and methods of preparation for
protein/peptide drug or bioactive agent delivery using a simple and
mild ionic-gelation method upon addition of a poly-.gamma.-glutamic
acid (.gamma.-PGA) solution (or other negatively charged component,
such as PGA-complexion conjugate) into regular molecular weight
chitosan (CS) solution. In one embodiment, the chitosan employed in
this invention are N-trimethyl chitosan (TMC), low MW-chitosan,
EDTA-chitosan, pegylated chitosan (PEG-chitosan),
mono-N-carboxymethyl chitosan (MCC), chitosan derivatives, and
combinations thereof. In one embodiment, the molecular weight of a
low-MW CS of the present invention is about 80 kDa or less,
preferably at about 40-50 kDa, adapted for adequate solubility at a
pH that maintains the bioactivity of protein and peptide drugs. It
is stipulated that a chitosan particle with about 30-50 kDa
molecular weight is kidney inert. The particle size and the zeta
potential value of the prepared nanoparticles are controlled by
their constituted compositions. The results obtained by the TEM
(transmission electron microscopy) and AFM (atomic force
microscopy) examinations showed that the morphology of the prepared
nanoparticles is generally spherical or spheroidal in shape.
[0011] Some aspects of the invention relate to a method of
enhancing epithelial permeation (for example, intestinal or blood
brain paracellular transport) configured for delivering at least
one bioactive agent, comprising administering nanoparticles
composed of .gamma.-PGA and chitosan, in an animal subject.
Administering the nanoparticles may be via oral administration,
intranasal absorption, subcutaneous injection or injection into a
blood vessel. In one embodiment, the chitosan dominates on the
surface of the nanoparticles as shell substrate and the negatively
charged .gamma.-PGA or other suitable component, with chitosan
present, as core substrate. In another embodiment, a substantial
surface of the nanoparticles is characterized with a positive
charge. In a further embodiment, the nanoparticles of the present
invention comprise at least one positively charged shell substrate
and at least one negatively charged core substrate. In one
embodiment, all of the negatively charged core substrate conjugates
with a portion of the positively charged shell substrate in the
core portion so to maintain a substantially zero-charged (neutral)
core. In one embodiment, at least one bioactive agent or protein
drug is conjugated with the negatively charged core substrate or
the substantially zero-charged (neutral) core.
[0012] In a further embodiment, the nanoparticles have a mean
particle size between about 50 and 500 nanometers, preferably
between about 100 and 300 nanometers, and most preferably between
about 100 and 200 nanometers.
[0013] In some embodiments, the nanoparticles are loaded with a
therapeutically effective amount of at least one bioactive agent,
wherein the bioactive agent is selected from the group consisting
of proteins, peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, oxygen-enriching agent,
oxygen-containing agent, anti-epileptic drug, and anti-inflammatory
drugs. The anti-epileptic drug may include Neurontin (gabapentin, a
gamma-aminobutyric acid analog), Lamictal (lamotrigine, shown to
act at voltage-sensitive sodium channels, stabilizing neural
membranes and inhibiting the release of excitatory neural
transmitters), Febatol (felbamate, shown to have weak inhibitory
effects on GABA receptor binding sites), Topamax (topiramate, has a
novel chemical structure derived from D-fructose that blocks
voltage-sensitive sodium channels, enhances the activity of GABA,
an inhibitory neurotransmitter, and blocks the action of glutamate,
an excitatory neurotransmitter), and/or Cerebyx (fosphenyloin, a
phenyloin precursor that is rapidly converted after parenteral
administration).
[0014] Further, the bioactive agent may be selected from the group
consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine,
enkephalin, tyrotropin releasing hormone, follicle stimulating
hormone, luteinizing hormone, vasopressin and vasopressin analogs,
catalase, superoxide dismutase, interleukin-11, interferon, colony
stimulating factor, tumor necrosis factor, tumor necrosis factor
inhibitor, and melanocyte-stimulating hormone. Interleukin eleven
(IL-11) is a thrombopoietic growth factor that directly stimulates
the proliferation of hematopoietic stem cells and megakaryocyte
progenitor cells and induces megakaryocyte maturation resulting in
increased platelet production (Oprelvekin.RTM.). In one preferred
embodiment, the bioactive agent is an Alzheimer antagonist.
[0015] Some aspects of the invention relate to an oral dose of
nanoparticles that effectively enhance epithelial permeation or
paracellular transport comprising .gamma.-PGA or .alpha.-PGA (or
other PGA derivatives, such as PGA-complexone conjugate that is
negatively charged) and low molecular weight chitosan, wherein the
chitosan dominates on a surface of the nanoparticles. Some aspects
of the invention relate to an oral dose of nanoparticles that
effectively enhance epithelial permeation (e.g., intestinal or
blood brain paracellular transport) comprising a negative
component, such as .gamma.-PGA, .alpha.-PGA, heparin, or heparan
sulfate, in the core, and low molecular weight chitosan dominating
on the surface of the nanoparticles with positive surface charges.
The core substrate may be selected from the group consisting of
heparin, heparin analogs, low molecular weight heparin,
glycosaminoglycans, and alginate, whereas the bioactive agent is
selected from the group consisting of chondroitin sulfate,
hyaluronic acid, growth factor and protein with a pharmaceutically
effective amount.
[0016] In a further embodiment, the nanoparticles comprise at least
one bioactive agent, such as insulin, insulin analog, Alzheimer's
disease antagonist, Parkison's disease antagonist, or other
protein/peptide. The bioactive agent for treating Alzheimer's
disease may include memantine hydrochloride (Axura.RTM. by Merz
Pharmaceuticals), donepezil hydrochloride (Aricept.RTM. by Eisai
Co. Ltd.), rivastigmine tartrate (Exelon.RTM. by Novartis),
galantamine hydrochloride (Reminyl.RTM. by Johnson & Johnson),
or tacrine hydrochloride (Cognex.RTM. by Parke Davis). Examples of
insulin or insulin analog products include, but not limited to,
Humulin.RTM. (by Eli Lilly), Humalog.RTM. (by Eli Lilly) and
Lantus.RTM. (by Aventis).
[0017] Some aspects of the invention provide a dose of
nanoparticles that enhance epithelial permeation, intestinal
permeation, or blood brain paracellular transport. Each
nanoparticle comprises three components; the first component of at
least one bioactive agent, a second component of low molecular
weight chitosan that is positively charged and a third component
that is negatively charged, wherein the second component dominates
on a surface of the nanoparticle. In one embodiment, the third
component is .gamma.-PGA, .alpha.-PGA, derivatives (such as
PGA-complexone conjugate and the like), salts of PGA, or
combinations thereof, heparin or alginate. In another embodiment,
the first component comprises insulin at a concentration range of
0.075 to 0.091 mg/ml, the second component at a concentration range
of 0.67 to 0.83 mg/ml, and the third component comprises
.gamma.-PGA at a concentration range of 0.150 to 0.184 mg/ml. The
at least one bioactive agent may comprise an antagonist for
Alzheimer's disease or for treatment of Alzheimer's disease
selected from the group consisting of memantine hydrochloride,
donepezil hydrochloride, rivastigmine tartrate, galantamine
hydrochloride, and tacrine hydrochloride. In a further embodiment,
the at least one bioactive agent is insulin or insulin analog. In
still another embodiment, the at least one bioactive agent is
selected from the group consisting of proteins, peptides,
nucleosides, nucleotides, antiviral agents, antineoplastic agents,
antibiotics, oxygen-enriching agent, oxygen-containing agent,
calcitonin, vancomycin, and anti-inflammatory drugs.
[0018] Some aspects of the invention provide a dose of
nanoparticles that enhance permeation, wherein the nanoparticles
are further encapsulated in a capsule or hard-cap capsule. In one
embodiment, the nanoparticles are freeze-dried. In one embodiment,
the interior surface of the capsule is treated to be lipophilic or
hydrophobic so to keep the enclosed ingredients or nanoparticles
intact or passive to the interior surface. In another embodiment,
the interior surface or the exterior surface of the capsules is
enteric-coated or treated with an enteric coating polymer.
[0019] Some aspects of the invention provide a method of enhancing
epithelial permeation comprising administering a dose of
nanoparticles, wherein each nanoparticle comprises a first
component of at least one bioactive agent, a second component of
low molecular weight chitosan, and a third component that is
negatively charged, wherein the second component dominates on a
surface of the nanoparticle. In one embodiment, the step of
administering the dose of nanoparticles is via oral administration
for enhancing epithelial permeation or intestinal paracellular
transport. In another embodiment, the step of administering the
dose of nanoparticles is via intravenous administration or
injection to a blood vessel for enhancing blood brain paracellular
transport or reducing the blood-brain barrier (BBB). In another
embodiment, the step of administering the nanoparticles is via
subcutaneous injection, intramuscular injection, or intranasal
spraying.
[0020] In one embodiment, the orally administered
insulin-containing nanoparticles comprise an effective dosage
amount of the insulin to treat the diabetes between about 5 units
to 95 units insulin, preferably between about 15 units to 45 units,
per kilogram body weight of the animal subject. In a further
embodiment, the insulin-containing nanoparticle comprises a trace
amount of zinc or calcium, or is treated with an enteric
coating.
[0021] In one embodiment, the bioactive agent-containing
nanoparticles further comprise at least one permeation enhancer,
wherein the permeation enhancer may be selected from the group
consisting of Ca.sup.2+ chelators, bile salts, anionic surfactants,
medium-chain fatty acids, phosphate esters, and the like. In
another embodiment, the nanoparticles and the permeation enhancer
are co-encapsulated in a capsule or are encapsulated separately in
two sets of capsules for co-administration.
[0022] Some aspects of the invention provide a method of treating
Alzheimer's diseases of an animal subject comprising intravenously
administering bioactive nanoparticles with an effective dosage to
treat the Alzheimer's diseases, wherein the bioactive nanoparticles
comprises a positively charged shell substrate, a negatively
charged or substantially neutral-charged core substrate, and at
least one bioactive agent for treating Alzheimer's disease, wherein
at least one bioactive agent is selected from the group consisting
of memantine hydrochloride, donepezil hydrochloride, rivastigmine
tartrate, galantamine hydrochloride, and tacrine hydrochloride.
[0023] In one embodiment, the effective treatment of the
Alzheimer's diseases comprises administering at least one bioactive
agent for treating Alzheimer's diseases at about 10 mg to 40 mg per
day over a period of one month to one year or longer. In another
embodiment, at least a portion of the shell substrate is
crosslinked, preferably at a degree of crosslinking less than about
50%, or most preferably between about 1% and 20%.
[0024] One aspect of the invention provides a pharmaceutical
composition of nanoparticles, wherein the nanoparticles may be
freeze-dried to form solid dried nanoparticles. The dried
nanoparticles may be loaded in a capsule (such as a two-part hard
gelatin capsule) or a tablet, which may be further enterically
coated, for oral administration in an animal subject. The
freeze-dried nanoparticles can be rehydrated in a solution or by
contacting body fluid as to revert to wet nanoparticles having
positive surface charge with substantially the properties of the
pre-lyophilized nanoparticles. In one embodiment, nanoparticles may
be mixed with trehalose or with hexan-1,2,3,4,5,6-hexyl in a
freeze-drying process. In one embodiment, the interior surface of
the capsule is treated to be lipophilic or hydrophobic. In another
embodiment, the exterior surface of the capsule is enteric-coated
or treated with an enteric coating polymer.
[0025] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles that enhance epithelial permeation or
paracellular transport, each nanoparticle comprising a shell
component and a core component, wherein at least a portion of the
shell component comprises chitosan and wherein the core component
is comprised of MgSO.sub.4, sodium tripolyphosphate, at least one
bioactive agent, and a negatively charged compound, wherein a
substantial portion of the negatively charged compound is
electrostatically bound to the chitosan.
[0026] Some aspects of the invention provide an orally deliverable
capsule to an animal subject comprising: (a) an empty capsule; and
(b) bioactive nanoparticles loaded within the empty capsule,
wherein the nanoparticles comprise a shell substrate of chitosan, a
negatively charged or substantially neutral-charged core substrate,
and (c) at least one bioactive agent. In one embodiment, the empty
capsule comprises a two-part hard gelatin capsule. In another
embodiment, the capsule is treated with an enteric coating polymer.
In one embodiment, the interior surface of the capsule may be
treated with hydrophobic or enteric coating.
[0027] One object of the present invention is to provide a method
of manufacturing the orally deliverable capsule, the method
comprising the steps of: (a) providing an empty capsule; (b)
providing bioactive nanoparticles, wherein the nanoparticles
comprise a shell substrate of chitosan, a negatively charged or
substantially zero-charged core substrate, and at least one
bioactive agent; (c) freeze-drying the nanoparticles; and (d)
filling the freeze-dried bioactive nanoparticles into the empty
capsule, thereby producing an orally deliverable capsule. In one
embodiment, the bioactive nanoparticles further comprise zinc,
magnesium sulfate and TPP.
[0028] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles for oral administration in an animal
subject, the nanoparticles comprising a shell portion that is
dominated by positively charged chitosan, a core portion that
contains negatively charged substrate, wherein the negatively
charged substrate is at least partially neutralized with a portion
of the positively charged chitosan in the core portion, and at
least one bioactive agent loaded within the nanoparticles. In one
embodiment, the bioactive agent is a non-insulin exenatide, a
non-insulin pramlintide, GLP-1, GLP-1 analog, GLP-2, GLP-2 analog,
insulin, insulin analog, or combinations thereof. In one
embodiment, the nanoparticles are formed via a simple and mild
ionic-gelation method. Glucagon-like peptide-2 (GLP-2) is a
recently identified potent intestinotrophic factor. The effect of
GLP-2 treatment on intestinal epithelial barrier function in mice
shows that the glucagon-like peptide-2 enhances intestinal
epithelial barrier function of both transcellular and paracellular
pathways in the mouse. Nevertheless, glucagon-like peptide-2
reduces intestinal permeability but does not modify the onset of
Type 1 diabetes in the nonobese diabetic mouse.
[0029] In one embodiment, a surface of the nanoparticles of the
pharmaceutical composition of the present invention is
characterized with a positive surface charge, wherein the
nanoparticles have a surface charge from about +15 mV to about +50
mV. In another embodiment, the nanoparticles have a mean particle
size between about 50 and 400 nanometers. In still another
embodiment, at least a portion of the shell portion of the
nanoparticles is crosslinked. In a further embodiment, the
nanoparticles are in a form of freeze-dried powder. In one
embodiment, the nanoparticles of the pharmaceutical composition of
the present invention further comprise iron, zinc, calcium,
magnesium sulfate and TPP.
[0030] Some aspects of the invention provide a method of delivering
a bioactive agent to blood circulation in an animal subject,
comprising: (a) providing nanoparticles according to the
pharmaceutical composition of the present invention, wherein the
nanoparticles are formed via a simple and mild ionic-gelation
method; (b) administering the nanoparticles orally toward an
intestine of the animal subject; (c) urging the nanoparticles to be
absorbed onto a surface of an epithelial membrane of the intestine;
(d) permeating bioactive agent to pass through an epithelial
barrier of the intestine; and (e) releasing the bioactive agent
into the blood circulation. In one embodiment, the bioactive agent
is selected from the group consisting of exenatide, pramlintide,
insulin, insulin analog, and combinations thereof.
[0031] Some aspects of the invention provide a method of delivering
a bioactive agent to an animal subject orally, the method
comprising formulating nanoparticles containing bioactive agent
according to the principles of the present disclosure, wherein the
nanoparticles are suspended in liquid. In one embodiment, the
liquid with nanoparticles containing bioactive agent is served as a
sport drink or energy drink. In one embodiment, the bioactive agent
is an oxygen-enriching agent or oxygen-containing agent (such as
hemoglobin). In another embodiment, the bioactive agent is an
energy-enhancing agent, such as CoQ.sub.10. Coenzyme Q.sub.10 (also
known as ubiquinone, ubidecarenone, coenzyme Q, and abbreviated at
times to CoQ.sub.10, CoQ, Q10, or Q) is a benzoquinone, where Q
refers to the quinone chemical group, and 10 refers to the
isoprenyl chemical subunits. This oil-soluble vitamin-like
substance is present in most eukaryotic cells, primarily in the
mitochondria. It is a component of the electron transport chain and
participates in aerobic cellular respiration, generating energy in
the form of ATP. Ninety-five percent of the human body's energy is
generated this way. Therefore, those organs with the highest energy
requirements--such as the heart and the liver--have the highest
CoQ.sub.10 concentrations or requirement.
[0032] Some aspects of the invention provide a method of reducing
inflammatory response caused by tumor necrosis factor in an animal
subject, the method comprising orally administering nanoparticles
composed of a TNF inhibitor, chitosan, and a core substrate of
poly(glutamic acid) or heparin. In one embodiment, the TNF
inhibitor is a monoclonal antibody. In another embodiment, the TNF
inhibitor is infliximab or adalimumab. In one embodiment, the TNF
inhibitor is a circulating receptor fusion protein. In another
embodiment, the TNF inhibitor is etanercept.
[0033] In one embodiment, the chitosan of the nanoparticles has a
molecular weight about 80 kDa or less. In another embodiment, the
chitosan is N-trimethyl chitosan or chitosan derivatives, such as
EDTA-chitosan. In still another embodiment, the poly(glutamic acid)
of the nanoparticles is .gamma.-PGA, .alpha.-PGA, PGA-complexone
conjugate, derivatives of PGA, salts of PGA, or combinations
thereof. In one embodiment, the nanoparticles have a mean particle
size between about 50 and 400 nanometers.
[0034] Some aspects of the invention relate to a method of treating
infections caused by microorganisms in an animal subject, the
method comprising administering nanoparticles composed of an
antibiotic, chitosan, and a core portion of negatively charged
substrate, wherein a surface of the nanoparticles is dominated by
the chitosan.
[0035] In one embodiment, the antibiotic is selected from the group
consisting of vancomycin, glycylcycline antibiotics (such as
Tigecycline), lincosamide antibiotics (such as Lincomycin),
beta-lactam antibiotic (such as Penicillin, Ampicillin, and
Piperacillin), bacteriophages, antitumor antibiotics, and
aminoglycoside antibiotics.
[0036] Some aspects of the invention provide a method of treating
diabetes in a subject, comprising co-administering, at least one
insulin anti-diabetic or non-insulin anti-diabetic drug and
enzyme-resistant PGA-complexone. In one embodiment, the non-insulin
anti-diabetic drug is metformin, osteocalcin, an insulin
secretagogue, a GLP-1 analog, a DPP-4 inhibitor, or selected from
the group consisting of alpha-glucosidase inhibitors, amylin
analog, sodium-glucose co-transporter type 2 (SGLT2) inhibitors,
benfluorex, and tolrestat.
[0037] Some aspects of the invention provide administering
bioactive nanoparticles to a subject with enhanced enzymatic
resistance to the bioactive agent inside the bioactive
nanoparticles, wherein the nanoparticles comprise a shell portion
that is dominated by positively charged chitosan, a core portion
that contains negatively charged substrate, wherein the negatively
charged substrate is at least partially neutralized with a portion
of the positively charged chitosan and at least one
enzyme-resistant agent. In one embodiment, the enzyme-resistant
agent is complexone, such as diethylene triamine pentaacetic acid
(DTPA) or ethylene diamine tetraacetic acid (EDTA), which may
conjugate with the chitosan substrate or the PGA substrate in the
nanoparticle formulation.
[0038] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles, the nanoparticles comprising a shell
portion that is dominated by positively charged chitosan, a core
portion that comprises one negatively charged substrate, wherein
the substrate is PGA-complexone conjugate, wherein the negatively
charged substrate is at least partially neutralized with a portion
of the positively charged chitosan in the core portion, and at
least one bioactive agent loaded within the nanoparticles. In one
embodiment, the pharmaceutical composition of nanoparticles further
comprises a pharmaceutically acceptable carrier, diluent, or
excipient.
[0039] In one embodiment, the nanoparticles are encapsulated in
capsules, wherein the capsules further comprise at least a
solubilizer, bubbling agent, emulsifier, pharmacopoeial excipients
or at least one permeation enhancer. In another embodiment, the
nanoparticles are freeze-dried, thereby the nanoparticles being in
a powder form.
[0040] Some aspects of the present invention provide a method of
treating an inflammatory bowel disease of an animal subject, the
method comprising administering bioactive nanoparticles to the
animal subject orally, wherein the bioactive nanoparticles consist
of at least one anti-inflammatory agent, positively charged
chitosan, optionally a zero-charge substance and a negatively
charged substrate, wherein a surface of the nanoparticles is
dominated by the positively charged chitosan. In one embodiment,
the negatively charged substrate is PGA that is selected from the
group consisting of a PGA-complexone conjugate, .gamma.-PGA,
.alpha.-PGA, derivatives of PGA, salts of PGA, or combinations
thereof. In another embodiment, the PGA-complexone conjugate is
PGA-DTPA that is chelated to gadolinium.
[0041] In one embodiment, the inflammatory bowel disease is a
Crohn's disease (autoimmune origin) or ulcerative colitis. In
another embodiment, the anti-inflammatory agent is selected from
the group consisting of mesalazine, prednisone, a TNF inhibitor,
azathioprine (Imuran), methotrexate, 6-mercaptopurine, nystatin,
antifungal agent, itraconazole, and fluconazole.
[0042] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles, the nanoparticles consisting of
positively charged chitosan, optionally a zero-charge substance or
bioactive agent, and a negatively charged substrate having
gadolinium (Gd) chelated to the negatively charged substrate,
wherein a surface of the nanoparticles is dominated by the
positively charged chitosan. In one embodiment, the chitosan is
N-trimethyl chitosan, EDTA-chitosan, low molecular weight chitosan,
PEG-chitosan, mono-N-carboxymethyl chitosan, chitosan derivatives,
or combinations thereof. In a preferred a ligand is attached a free
--NH.sub.3 group of the N-trimethyl chitosan. In another
embodiment, the ligand includes a substrate, inhibitor, activator,
or neurotransmitter, for example galactosamine.
[0043] In one embodiment, the negatively charged substrate of the
nanoparticles is a PGA-complexone conjugate, .gamma.-PGA,
.alpha.-PGA, derivatives of PGA, salts of PGA, or combinations
thereof. In a further embodiment, the PGA-complexone conjugate is
PGA-DTPA.
[0044] In one embodiment, the nanoparticles of the pharmaceutical
composition are formulated into a tablet, capsule, or pill
configuration, wherein the tablet, capsule, or pill is optionally
enterically coated (i.e., treated with an enteric coating polymer).
In one embodiment, the capsule further comprises a pharmaceutically
acceptable carrier, diluent, excipient, absorption enhancer, at
least a solubilizer, bubbling agent, or emulsifier. In a further
embodiment, the nanoparticles are freeze-dried, thereby the
nanoparticles being in a powder form.
[0045] In one embodiment, the nanoparticles are used in (or
characterized by) enhancing imaging contrast quality or property
during an imaging procedure. In another embodiment, the
nanoparticles are used in (or characterized by) cancer treatment
therapy. The nanoparticles may be administered to an animal subject
via an oral or parenteral route. The bioactive agent of the
nanoparticles is selected from the group consisting of an
anti-cancer drug, nystatin, antifungal agent, itraconazole,
fluconazole, mesalazine, prednisone, a TNF inhibitor, azathioprine
(Imuran), methotrexate, the 6-mercaptopurine. In one embodiment,
the zero-charge substance of the nanoparticles is a permeation
enhancer.
[0046] Some aspects of the invention provide a method of delivering
a bioactive agent to an animal subject with enhanced enzymatic
inhibition property, the method comprising co-administering a
PGA-complexone conjugate and the bioactive agent to the animal
subject orally. More particularly, a method of delivering a peptide
or protein drug to an animal subject with enhanced enzymatic
inhibition property or with mitigated enzyme activity, the method
comprising co-administering a PGA-complexone conjugate and the drug
to the animal subject orally. In one embodiment, the PGA-complexone
conjugate is PGA-DTPA (polyglutamic acid-diethylene triamine
pentaacetic acid).
[0047] Some aspects of the invention provide a method of
co-delivering a peptide or protein drug and a PGA-complexone
conjugate to an animal subject orally toward the gastrointestinal
tract, wherein the PGA-complexone conjugate and the drug are
formulated into a tablet, pill, or capsule configuration. In one
embodiment, the tablet, pill, or capsule is treated with an enteric
coating polymer. In a further embodiment, the capsule further
comprises a pharmaceutically acceptable carrier, diluent,
excipient, a solubilizer, bubbling agent, or emulsifier. In one
embodiment, the capsule further comprises at least one permeation
enhancer, wherein the permeation enhancer is selected from the
group consisting of Ca.sup.2+ chelators, bile salts, surfactants,
medium-chain fatty acids, phosphate esters, and chitosan. And the
chitosan may be selected from the group consisting of N-trimethyl
chitosan (TMC), low MW-chitosan, EDTA-chitosan, pegylated chitosan
(PEG-chitosan), mono-N-carboxymethyl chitosan (MCC), chitosan
derivatives, and combinations thereof.
[0048] Some aspects of the invention provide a method of
co-delivering a peptide or protein drug and a PGA-complexone
conjugate to an animal subject orally, wherein the drug is a
pegylated drug. In one embodiment, the drug is covalently attached
with polyethylene glycol polymer chains. In another embodiment, the
drug is an anti-diabetic drug or a drug for treating diabetes.
[0049] Some aspects of the invention provide a method for treating
disorders of a tight junction comprising delivering a nanoparticle
delivery system to the tight junction, wherein the nanoparticle
delivery system comprises nanoparticles or fragments thereof
according to a pharmaceutical composition disclosed. In one
embodiment, the bioactive agent is selected from the group
consisting of anti-epileptic drugs, anti-inflammatory drugs,
meningitis antagonist, and anti-oxidant. Some aspects of the
invention provide a method for treating disorders or diseases of a
tight junction comprising delivering a pharmaceutical composition
of nanoparticles to the tight junction, wherein the nanoparticles
consist of positively charged chitosan, a negatively charged
substrate, optionally a zero-charge compound, and at least one
bioactive agent for treating the disorders or diseases of the tight
junction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] Additional objects and features of the present invention
will become more apparent and the disclosure itself will be best
understood from the following Detailed Description of the Exemplary
Embodiments, when read with reference to the accompanying
drawings.
[0051] FIG. 1 shows GPC chromatograms of (a) standard-MW CS before
depolymerization and the low-MW CS after depolymerization; (b) the
purified .gamma.-PGA obtained from microbial fermentation.
[0052] FIG. 2 shows (a) FT-IR and (b) .sup.1H-NMR spectra of the
purified .gamma.-PGA obtained from microbial fermentation.
[0053] FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared
CS-.gamma.-PGA nanoparticles.
[0054] FIG. 4 shows (a) a TEM micrograph of the prepared
CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20% CS) and (b)
an AFM micrograph of the prepared CS-.gamma.-PGA nanoparticles
(0.01% .gamma.-PGA:0.01% CS).
[0055] FIG. 5 shows changes in particle size and zeta potential of
(a) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20% CS)
and (b) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.01%
CS) during storage for up to 6 weeks.
[0056] FIG. 6 shows effects of the prepared CS-.gamma.-PGA
nanoparticles on the TEER values of Caco-2 cell monolayers.
[0057] FIG. 7 shows fluorescence images (taken by an inversed
confocal laser scanning microscope) of 4 optical sections of a
Caco-2 cell monolayer that had been incubated with the
fCS-.gamma.-PGA nanoparticles with a positive surface charge (0.10%
.gamma.-PGA:0.20% CS) for (a) 20 min and (b) 60 min.
[0058] FIG. 8 shows an illustrative protein transport mechanism
through a cell layer, including transcellular transport and
paracelluler transport.
[0059] FIGS. 9 A-C show a schematic illustration of a paracellular
transport mechanism.
[0060] FIG. 10 shows a CS-.gamma.-PGA nanoparticle with chitosan
having positive surface charge.
[0061] FIG. 11 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan and .gamma.-PGA.
[0062] FIG. 12 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan as reference.
[0063] FIG. 13 shows the stability of insulin-loaded
nanoparticles.
[0064] FIG. 14 shows a representative in vitro study with an
insulin drug release profile in a pH-adjusted solution.
[0065] FIG. 15 shows the effect of insulin of orally administered
insulin-loaded nanoparticles on hypoglycemia in diabetic rats.
[0066] FIGS. 16 A-C show a proposed mechanism of nanoparticles
released from the enteric-coated capsules.
[0067] FIG. 17 shows the effect of orally administered
insulin-loaded nanoparticles on `glucose reduction %` in diabetic
rats, wherein the freeze-dried nanoparticles were loaded in an
enterically coated capsule upon delivery.
[0068] FIG. 18 shows insulin-loaded nanoparticles with a core
composition consisted of .gamma.-PGA, MgSO.sub.4, sodium
tripolyphosphate (TPP), and insulin.
[0069] FIG. 19 shows an in vivo subcutaneous study using insulin
injectables and insulin-containing nanoparticles.
[0070] FIG. 20 shows experimental data on enzyme inhibition study
with (.gamma.-PGA)-DTPA conjugate.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0071] The preferred embodiments of the present invention described
below relate particularly to the preparation of nanoparticles
composed of chitosan/poly-glutamic acid/insulin and their
permeability to enhance the intestinal or blood brain paracellular
permeation by opening the tight junctions between epithelial cells.
While the description sets forth various embodiment specific
details, it should be understood that the description is
illustrative only and should not be construed in any way as
limiting the invention. Furthermore, various applications of the
invention, and modifications thereto, which may occur to those who
are skilled in the art, are also encompassed by the general
concepts described below.
[0072] "Bioactive agent" herein is meant to include any agent that
may affect the recipient (an animal subject) after being
administered physically, physiologically, mentally, biochemically,
biologically, or other bodily functions in a positive or negative
manners. The `bioactive agent` may include, but not limited to,
drugs, protein, peptides, siRNA, enzymes, supplemental nutrients,
vitamins, other active agents.
[0073] .gamma.-PGA is a naturally occurring anionic homo-polyamide
that is made of L-glutamic acid units connected by amide linkages
between .alpha.-amino and .gamma.-carboxylic acid groups (Crit.
Rev. Biotechnol. 2001; 21:219-232). It is an exocellular polymer of
certain Bacillus species that is produced within cells via the TCA
cycle and is freely excreted into the fermentation broth. Its exact
biological role is not fully known, although it is likely that
.gamma.-PGA is linked to increasing the survival of producing
strains when exposed to environmental stresses. Because of its
water-solubility, biodegradability, edibility, and non-toxicity
toward humans and the environment, several applications of
.gamma.-PGA in food, cosmetics, medicine, and water treatment have
been investigated in the past few years.
Example No. 1
Materials and Methods of Nanoparticles Preparation
[0074] CS (MW .about.2.8.times.10.sup.5) with a degree of
deacetylation of approximately 85% was acquired from Challenge
Bioproducts Co. (Taichung, Taiwan). Acetic acid, cellulase (1.92
units/mg), fluorescein isothiocyanate (FITC), phosphate buffered
saline (PBS), periodic acid, sodium acetate, formaldehyde, bismuth
subnitrate, and Hanks balanced salt solution (HESS) were purchased
from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absolute
anhydrous and potassium sodium tartrate were obtained from Merck
(Darmstadt, Germany). Non-essential amino acid (NEAA) solution,
fetal bovine serum (FBS), gentamicin and trypsin-EDTA were acquired
from Gibco (Grand Island, N.Y.). Eagle's minimal essential medium
(MEM) was purchased from Bio West (Nuaille, France). All other
chemicals and reagents used were of analytical grade.
Example No. 2
Depolymerization of CS by Enzymatic Hydrolysis
[0075] Regular CS was treated with enzyme (cellulase) to produce
low-MW CS according to a method described by Qin et al. with some
modifications (Food Chem. 2004; 84:107-115). A solution of CS (20
g/l) was prepared by dissolving CS in 2% acetic acid. Care was
taken to ensure the total solubility of CS. Then, the CS solution
was introduced into a vessel and adjusted to the desired pH 5.0
with 2N aqueous NaOH. Subsequently, cellulase (0.1 g) was added
into the CS solution (100 ml) and continuously stirred at
37.degree. C. for 12 hours. Afterward, the depolymerized CS was
precipitated with aqueous NaOH at pH 7.0-7.2 and the precipitated
CS was washed three times with deionized water. The resulting
low-MW CS was lyophilized in a freeze dryer (Eyela Co. Ltd, Tokyo,
Japan).
[0076] The average molecular weight of the depolymerized CS was
determined by a gel permeation chromatography (GPC) system equipped
with a series of PL aquagel-OH columns (one Guard 8 .mu.m,
50.times.7.5 mm and two MIXED 8 .mu.m, 300.times.7.5 mm, PL
Laboratories, UK) and a refractive index (RI) detector (RI2000-F,
SFD, Torrance, Calif.). Polysaccharide standards (molecular weights
range from 180 to 788,000, Polymer Laboratories, UK) were used to
construct a calibration curve. The mobile phase contained 0.01M
NaH.sub.2PO.sub.4 and 0.5M NaNO.sub.3 and was brought to a pH of
2.0. The flow rate of the mobile phase was 1.0 ml/min, and the
columns and the RI detector cell were maintained at 30.degree.
C.
[0077] Factors limiting applications of most commercially available
CSs are their high molecular weight and corresponding high
viscosity and poor solubility at physiological pH ranges. Low-MW CS
overcomes these limitations and hence finds much wider applications
in diversified fields. It was suggested that low-MW CS be used as a
parenteral drug carrier due to its lower antigen effect (Eur. J.
Pharm. Biopharm. 2004; 57:101-105). Low-MW CS was used as a
non-viral gene delivery system and showed promising results (Int.
J. Pharm. 1999; 178:231-243). Other studies based on animal testing
showed the possibilities of low-MW CS for treatment of type 2
diabetes and gastric ulcer (Biol. Pharm. Bull. 2002; 25:188-192).
Several hydrolytic enzymes such as lysozyme, pectinase, cellulase,
bromelain, hemicellulase, lipase, papain and the like can be used
to depolymerize CS (Biochim. Biophys. Acta 1996; 1291:5-15;
Biochem. Eng. J. 2001; 7:85-88; Carbohydr. Res. 1992;
237:325-332).
[0078] FIG. 1a shows GPC chromatograms of both standard-MW (also
known as regular-MW) and low-MW CS. It is known that cellulase
catalyzes the cleavage of the glycosidic linkage in CS (Food Chem.
2004; 84:107-115). The low-MW CS used in the study was obtained by
precipitating the depolymerized CS solution with aqueous NaOH at pH
7.0-7.2. This low-MW CS had a MW of about 50 kDa (FIG. 1a). In a
preferred embodiment, the low molecular weight chitosan has a
molecular weight of less than about 40 kDa, but above 10 kDa. Other
forms of chitosan may also be applicable, including chitin,
chitosan oligosaccharides, and derivatives thereof.
[0079] It was observed that the obtained low-MW CS can be readily
dissolved in an aqueous solution at pH 6.0, while that before
depolymerization needs to be dissolved in an acetic acid solution
with a pH value about 4.0. Additionally, it was found that with the
low-MW CS, the prepared nanoparticles had a significantly smaller
size with a narrower distribution than their counterparts prepared
with the high-MW (also known as standard-MW) CS (before
depolymerization), due to its lower viscosity. As an example, upon
adding a 0.10% .gamma.-PGA aqueous solution into a 0.20% high-MW CS
solution (viscosity 5.73.+-.0.08 cp, measured by a viscometer), the
mean particle size of the prepared nanoparticles was 878.3.+-.28.4
nm with a polydispersity index of 1.0, whereas adding a 0.10%
.gamma.-PGA aqueous solution into the low-MW CS solution (viscosity
1.29.+-.0.02 cp) formed nanoparticles with a mean particle size of
218.1.+-.4.1 nm with a polydispersity index of 0.3 (n=5).
[0080] The purified .gamma.-PGA used in forming nanoparticles of
the present invention was analyzed by GPC, .sup.1H-NMR, and FT-IR.
As analyzed by GPC (FIG. 1b), the purified .gamma.-PGA had a MW of
about 160 kDa. In the FT-IR spectrum (FIG. 2a), a characteristic
peak at 1615 cm.sup.-1 for the associated carboxylic acid salt
(--COO.sup.- antisymmetric stretch) on .gamma.-PGA was observed.
The characteristic absorption due to C.dbd.O in secondary amides
(amide I band) was overlapped by the characteristic peak of
--COO.sup.-. Additionally, the characteristic peak observed at 3400
cm.sup.-1 was the N--H stretch of .gamma.-PGA. In the .sup.1H-NMR
spectrum (FIG. 2b), six chief signals were observed at 1.73 and
1.94 ppm (.beta.-CH.sub.2), 2.19 ppm (.gamma.-CH.sub.2), 4.14 ppm
(.alpha.-CH), 8.15 ppm (amide), and 12.58 ppm (COOH). These results
indicated that the observed FT-IR and .sup.1H-NMR spectra
correspond well to those expected for .gamma.-PGA. Additionally,
the fermented product after purification showed no detected
macromolecular impurities by the .sup.1H-NMR analysis, suggesting
that the obtained white power of .gamma.-PGA is highly pure.
Example No. 3
Preparation of the CS-.gamma. PGA Nanoparticles
[0081] Nanoparticles were obtained upon addition of .gamma.-PGA
aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at varying
concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% by w/v) under
magnetic stirring at room temperature. Nanoparticles were collected
by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were
discarded and nanoparticles were resuspended in deionized water for
further studies. The nanoparticles thus obtained via the simple and
mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge and a
narrow polydispersity index. FT-IR was used to analyze peak
variations of amino groups of low-MW CS and carboxylic acid salts
of .gamma.-PGA in the CS-.gamma.-PGA nanoparticles.
[0082] As stated, nanoparticles were obtained instantaneously upon
the addition of a .gamma.-PGA aqueous solution (pH 7.4) into a
low-MW CS aqueous solution (pH 6.0) under magnetic stirring at room
temperature. FIG. 3 shows the FT-IR spectra of the low-MW CS and
the CS-.gamma.-PGA nanoparticles. As shown in the spectrum of CS,
the characteristic peak observed at 1563 cm.sup.-1 was the
protonated amino group (--NH.sub.3.sup.+ deformation) on CS. In the
spectrum of CS-.gamma.-PGA complex, the characteristic peak at 1615
cm.sup.-1 for --COO.sup.- on .gamma.-PGA disappeared and a new peak
at 1586 cm.sup.-1 appeared, while the characteristic peak of
--NH.sub.3.sup.+ deformation on CS at 1563 cm.sup.-1 shifted to
1555 cm.sup.-1. These observations are attributed to the
electrostatic interaction between the negatively charged carboxylic
acid salts (--COO.sup.-) on .gamma.-PGA and the positively charged
amino groups (--NH.sub.3.sup.+) on CS (Int. J. Pharm. 2003;
250:215-226). The electrostatic interaction between the two
polyelectrolytes (.gamma.-PGA and CS) instantaneously induced the
formation of long hydrophobic segments (or at least segments with a
high density of neutral ion-pairs), and thus resulted in highly
neutralized complexes that segregated into colloidal nanoparticles
(Langmuir. 2004; 20:7766-7778).
[0083] The particle sizes and the zeta potential values of the
CS-.gamma.-PGA nanoparticles, prepared at varying concentrations of
the .gamma.-PGA and CS, were determined and the results are shown
in Tables 1a and 1b. FIG. 10 shows a CS-.gamma.-PGA nanoparticle
with chitosan having positive surface charge. It was found that the
particle size and the zeta potential value of the prepared
nanoparticles were mainly determined by the relative amount of the
local concentration of the .gamma.-PGA in the added solution to the
surrounding concentration of CS in the sink solution. At a fixed
concentration of CS, an increase in the .gamma.-PGA concentration
allowed .gamma.-PGA molecules to interact with more CS molecules,
and thus formed a larger size of nanoparticles (Table 1a,
p<0.05). When the amount of CS molecules exceeded that of local
.gamma.-PGA molecules, some of the excess CS molecules were
entangled onto the surfaces of CS-.gamma.-PGA nanoparticles.
[0084] Thus, the resulting nanoparticles may display a structure of
a neutral polyelectrolyte-complex core surrounded by a positively
charged CS shell (Table 1b) ensuring a colloidal stabilization
(Langmuir. 2004; 20:7766-7778). In contrast, as the amount of local
.gamma.-PGA molecules sufficiently exceeded that of surrounding CS
molecules, the formed nanoparticles had .gamma.-PGA exposed on the
surfaces and thus had a negative charge of zeta potential.
Therefore, the particle size and the zeta potential value of the
prepared CS-.gamma.-PGA nanoparticles can be controlled by their
constituted compositions. The results obtained by the TEM and AFM
examinations showed that the morphology of the prepared
nanoparticles was spherical in shape with a smooth surface (FIGS.
4a and 4b). Some aspects of the invention relate to nanoparticles
having a mean particle size between about 50 and 400 nanometers,
and preferably between about 100 and 300 nanometers.
[0085] The morphology of the nanoparticles is spherical in shape
with a smooth surface at any pH between 2.5 and 6.6. In one
embodiment, the stability of the nanoparticles of the present
invention at a low pH around 2.5 enables the nanoparticles to be
intact when exposed to the acidic medium in the stomach.
[0086] Two representative groups of the prepared nanoparticles were
selected for the stability study: one with a positive surface
charge (0.10% .gamma.-PGA:0.20% CS) and the other with a negative
surface charge (0.10% .gamma.-PGA:0.01% CS). FIG. 5 shows changes
in particle size (.box-solid., mean diameter) and zeta potential (
) of (a) the CS-.gamma.-PGA nanoparticles (0.10% .gamma.-PGA:0.20%
CS) and (b) the CS-.gamma.-PGA nanoparticles (0.10%
.gamma.-PGA:0.01% CS) during storage up to 6 weeks. It was found
that neither aggregation nor precipitation of nanoparticles was
observed during storage up to 6 weeks, as a result of the
electrostatic repulsion between the positively charged
CS-.gamma.-PGA nanoparticles (for the former group) and the
negatively charged CS-.gamma.-PGA nanoparticles (for the latter
group).
[0087] Additionally, changes in particle size and zeta potential of
the nanoparticles were minimal for both studied groups (FIGS. 5a
and 5b). These results demonstrated that the prepared nanoparticles
suspended in deionized water were stable during storage.
[0088] In a further study, NPs were self-assembled instantaneously
upon addition of an aqueous .gamma.-PGA into an aqueous TMC
(N-trimethyl chitosan) having a TMC/.gamma.-PGA weight ratio of 6:1
under magnetic stirring at room temperature. Other chitosan
derivative, such as mono-N-carboxymethyl chitosan (MCC), has also
been useful in self-assembled nanoparticle formation. The chemical
formulas of chitosan, N-trimethyl chitosan, and MCC are shown
below:
##STR00001##
TABLE-US-00001 TABLE 1a Effects of concentrations of .gamma.-PGA
and CS on the particle sizes of the prepared CS-.gamma.-PGA
nanoparticles Mean Particle Size (nm, n = 5) CS .gamma.-PGA 0.01%
.sup.a) 0.05% 0.10% 0.15% 0.20% 0.01% .sup.b) 79.0 .+-. 3.0 103.1
.+-. 4.6 96.7 .+-. 1.9 103.6 .+-. 1.9 140.5 .+-. 2.0 0.05% 157.4
.+-. 1.7 120.8 .+-. 3.9 144.5 .+-. 2.4 106.2 .+-. 3.8 165.4 .+-.
1.7 0.10% 202.2 .+-. 3.1 232.6 .+-. 1.2 161.0 .+-. 1.8 143.7 .+-.
2.7 218.1 .+-. 4.1 0.15% 277.7 .+-. 3.2 264.9 .+-. 2.1 188.6 .+-.
2.9 178.0 .+-. 2.2 301.1 .+-. 6.4 0.20% 284.1 .+-. 2.1 402.2 .+-.
4.0 .tangle-solidup. 225.5 .+-. 3.1 365.5 .+-. 5.1 .sup.a)
concentration of CS (by w/v) .sup.b) concentration of .gamma.-PGA
(by w/v) .tangle-solidup. precipitation of aggregates was
observed
TABLE-US-00002 TABLE 1b Effects of concentrations of .gamma.-PGA
and CS on the zeta potential values of the prepared CS-.gamma.-PGA
nanoparticles. Zeta Potential (mV, n = 5) CS .gamma.-PGA 0.01%
.sup.a) 0.05% 0.10% 0.15% 0.20% 0.01% .sup.b) 15.4 .+-. 0.3 22.8
.+-. 0.5 19.8 .+-. 1.5 16.5 .+-. 1.4 17.2 .+-. 1.6 0.05% -32.7 .+-.
0.7 23.7 .+-. 1.7 27.6 .+-. 0.7 20.3 .+-. 0.8 19.2 .+-. 0.6 0.10%
-33.1 .+-. 1.3 21.1 .+-. 1.6 20.3 .+-. 1.1 23.6 .+-. 0.9 24.7 .+-.
1.2 0.15% -33.2 .+-. 2.1 -21.9 .+-. 2.0 19.2 .+-. 0.4 16.9 .+-. 1.7
19.8 .+-. 0.3 0.20% -34.5 .+-. 0.5 -34.6 .+-. 0.3 .tangle-solidup.
14.6 .+-. 0.7 16.3 .+-. 0.7 .sup.a) concentration of CS (by w/v)
.sup.b) concentration of .gamma.-PGA (by w/v) .tangle-solidup.
precipitation of aggregates was observed
[0089] The amount of positively charged TMC significantly exceeded
that of negatively charged .gamma.-PGA; some of excessive TMC
molecules were entangled onto the surfaces of NPs, thus displaying
a positive surface charge (Table 2). The degree of quaternization
on TMC had little effects on the mean particle size and zeta
potential of NPs.
TABLE-US-00003 TABLE 2 Mean particle sizes, zeta potential values
and polydispersity indices of nanoparticles (NPs) self-assembled by
TMC polymers with different degrees of quaternization and
.gamma.-PGA (n = 5 batches). Mean Particle Zeta Potential
Polydispersity Size (nm) (mV) Index CS/.gamma.-PGA NPs 104.1 .+-.
1.2 36.2 .+-. 2.5 0.11 .+-. 0.02 TMC25/.gamma.-PGA NPs 101.3 .+-.
3.1 30.9 .+-. 2.1 0.13 .+-. 0.04 TMC40/.gamma.-PGA NPs 106.3 .+-.
2.3 32.3 .+-. 2.1 0.15 .+-. 0.14 TMC55/.gamma.-PGA NPs 114.6 .+-.
2.3 30.6 .+-. 3.8 0.12 .+-. 0.03 TMC: N-trimethyl chitosan; CS:
chitosan; .gamma.-PGA: poly(.gamma.-glutamic acid).
Example No. 4
Caco-2 Cell Cultures and TEER Measurements
[0090] Caco-2 cells were seeded on the tissue-culture-treated
polycarbonate filters (diameter 24.5 mm, growth area 4.7 cm.sup.2)
in Costar Transwell 6 wells/plates (Corning Costar Corp., NY) at a
seeding density of 3.times.10.sup.5 cells/insert. MEM (pH 7.4)
supplemented with 20% FBS, 1% NEAA, and 40 .mu.g/ml
antibiotic-gentamicin was used as the culture medium, and added to
both the donor and acceptor compartments. The medium was replaced
every 48 hours for the first 6 days and every 24 hours thereafter.
The cultures were kept in an atmosphere of 95% air and 5% CO.sub.2
at 37.degree. C. and were used for the paracellular transport
experiments 18-21 days after seeding (TEER values in the range of
600-800 .OMEGA.cm.sup.2).
[0091] The intercellular tight junction is one of the major
barriers to the paracellular transport of macromolecules (J.
Control. Release 1996; 39:131-138; J. Control. Release 1998;
51:35-46). Trans-epithelial ion transport is contemplated to be a
good indication of the tightness of the junctions between cells and
therefore evaluated by measuring TEER of Caco-2 cell monolayers in
the study. It was reported that the measurement of TEER can be used
to predict the paracellular transport of hydrophilic molecules
(Eur. J. Pharm. Biopharm. 2004; 58:225-235). When the tight
junctions open, the TEER value will be reduced due to the water and
ion passage through the paracellular route. Caco-2 cell monolayers
have been widely used as an in vitro model to evaluate the
intestinal paracellular permeability of macromolecules.
[0092] Effects of the prepared CS-.gamma.-PGA nanoparticles on the
TEER values of Caco-2 cell monolayers are shown in FIG. 6. As
shown, the prepared nanoparticles with a positive surface charge
(CS dominated on the surface, 0.01% .gamma.-PGA:0.05% CS, 0.10%
.gamma.-PGA:0.2% CS, and 0.20% .gamma.-PGA:0.20% CS) were able to
reduce the values of TEER of Caco-2 cell monolayers significantly
(p<0.05). After a 2-hour incubation with these nanoparticles,
the TEER values of Caco-2 cell monolayers were reduced to about 50%
of their initial values as compared to the control group (without
addition of nanoparticles in the transport media). This indicated
that the nanoparticles with CS dominated on the surfaces could
effectively open or loosen the tight junctions between Caco-2
cells, resulting in a decrease in the TEER values. It was reported
that interaction of the positively charged amino groups of CS with
the negatively charged sites on cell surfaces and tight junctions
induces a redistribution of F-actin and the tight junction's
protein ZO-1, which accompanies the increased paracellular
permeability (Drug Deliv. Rev. 2001; 50:S91-S101). It is suggested
that an interaction between chitosan and the tight junction protein
ZO-1, leads to its translocation to the cytoskeleton.
[0093] After removal of the incubated nanoparticles, a gradual
increase in TEER values was noticed. This phenomenon indicated that
the intercellular tight junctions of Caco-2 cell monolayers started
to recover gradually; however, the TEER values did not recover to
their initial values (FIG. 6). Kotze et al. reported that complete
removal of a CS-derived polymer, without damaging the cultured
cells, was difficult due to the highly adhesive feature of CS
(Pharm. Res. 1997; 14:1197-1202). This might be the reason why the
TEER values did not recover to their initial values. In contrast,
the TEER values of Caco-2 cell monolayers incubated with the
nanoparticles with a negative surface charge (.gamma.-PGA dominated
on the surface, 0.10% .gamma.-PGA:0.01% CS and 0.20%
.gamma.-PGA:0.01% CS, FIG. 6) showed no significant differences as
compared to the control group (p>0.05). This indicated that
.gamma.-PGA does not have any effects on the opening of the
intercellular tight junctions.
[0094] FIG. 8 shows an illustrative protein transport mechanism
through a cellular layer, including transcellular transport and
paracelluler transport. FIG. 9 shows a schematic illustration of a
paracellular transport mechanism. The transcellular protein or
peptide transport may be either an active transport or a passive
transport mode whereas the paracellular transport is basically a
passive mode. Ward et al. reported and reviewed current knowledge
regarding the physiological regulation of tight junctions and
paracellular permeability (PSTT 2000; 3:346-358). Chitosan as
nanoparticle vehicles for oral delivery of protein drugs avoids the
enzymatic inactivation in the gastrointestinal conduit. The
chitosan component of the present nanoparticles has a special
feature of adhering to the mucosal surface and transiently opening
the tight junctions between epithelial cells; that is, loosening
the tightness of the tight junctions.
[0095] FIG. 9(A) shows that after feeding nanoparticles (NPs)
orally, NPs adhere and infiltrate into the mucus layer of the
epithelial cells. FIG. 9(B) illustrates that the infiltrated NPs
transiently and reversibly loosen tight junctions (TJs) while
becoming unstable and disintegrated to release insulin or another
entrapped agent. FIG. 9(c) shows that the released insulin or agent
permeates through the paracellular pathway into the blood stream.
Chitosan (CS), a nontoxic, soft-tissue compatible, cationic
polysaccharide has special features of adhering to the mucosal
surface; CS is able to transiently and reversibly widen/loosen TJs
between epithelial cells. Therefore, a nanoparticle shelled with
positively charged chitosan is able to function as a drug delivery
vehicle to carry the payload (for example, an antagonist for
treatment of disorders or diseases of a tight junction) to the TJ
site. The TJ width in the small intestine has been demonstrated to
be less than 1 nm. It is also known that TJs `opened` by absorption
enhancers are less than 20 nm wide (Nanotechnology 2007; 18:1-11).
The term "opened" herein means that any substance less than 20 nm
in the close-proximity might have the chance to pass through. TJs
are the principal barrier to passive movement of fluid,
electrolytes, macromolecules and cells through the paracellular
pathway.
[0096] It was suggested that the electrostatic interaction between
the positively charged CS and the negatively charged sites of ZO-1
proteins on cell surfaces at TJ induces a redistribution of
cellular F-actin as well as ZO-1's translocation to the
cytoskeleton, resulting in an increase in permeability. As
evidenced in FIG. 9, after adhering and infiltrating into the mucus
layer of the duodenum, the orally administered nanoparticles may
degrade due to the presence of distinct digestive enzymes in the
intestinal fluids. Additionally, the pH environment may become
neutral while the nanoparticles were infiltrating into the mucosa
layer and approaching the intestinal epithelial cells. This further
leads to the collapse of nanoparticles due to the change in the
exposed pH environment. The dissociated CS from the
degraded/collapsed nanoparticles was then able to interact and
modulate the function of ZO-1 proteins between epithelial cells
(Nanotechnology 2007; 18:1-11). ZO-1 proteins are thought to be a
linkage molecule between occludin and F-actin cytoskeleton as well
as play important roles in the rearrangement of cell-cell contacts
at TJs.
Example No. 5
fCS-.gamma.-PGA Nanoparticle Preparation and CLSM Visualization
[0097] Fluorescence (FITC)-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA)
nanoparticles were prepared for the confocal laser scanning
microscopy (CLSM) study. The nanoparticles of the present invention
display a structure of a neutral polyelectrolyte-complex core
surrounded by a positively charged chitosan shell. Synthesis of the
FITC-labeled low-MW CS (fCS) was based on the reaction between the
isothiocyanate group of FITC and the primary amino groups of CS as
reported in the literature (Pharm. Res. 2003; 20:1812-1819).
Briefly, 100 mg of FITC in 150 ml of dehydrated methanol were added
to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hours of
reaction in the dark at ambient conditions, fCS was precipitated by
raising the pH to about 8-9 with 0.5M NaOH. To remove the
unconjugated FITC, the precipitate was subjected to repeated cycles
of washing and centrifugation (40,000.times.g for 10 min) until no
fluorescence was detected in the supernatant. The fCS dissolved in
80 ml of 0.1M acetic acid, then dialyzed for 3 days in the dark
against 5 liters of distilled water, with the water replaced on a
daily basis. The resulting fCS was lyophilized in a freeze dryer.
The fCS-.gamma.-PGA nanoparticles were prepared as per the
procedure described in Example No. 3.
[0098] Afterward, the transport medium containing fCS-.gamma.-PGA
nanoparticles (0.2 mg/ml) was introduced into the donor compartment
of Caco-2 cells, which were pre-cultured on the transwell for 18-21
days. The experimental temperature was maintained at 37.degree. C.
by a temperature control system (DH-35 Culture Dish Heater, Warner
Instruments Inc., Hamden, Conn.). After incubation at specific time
intervals, test samples were aspirated. The cells were then washed
twice with pre-warmed PBS solution before they were fixed in 3.7%
paraformaldehyde (Pharm. Res. 2003; 20:1812-1819). Cells were
examined under an inversed CLSM (TCS SL, Leica, Germany). The
fluorescence images were observed using an argon laser (excitation
at 488 nm, emission collected at a range of 510-540 nm).
[0099] A CLSM was used to visualize the transport of the
fluorescence-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA) nanoparticles
across the Caco-2 cell monolayers. This non-invasive method allows
for optical sectioning and imaging of the transport pathways across
the Caco-2 cell monolayers, without disrupting their structures (J.
Control. Release 1996; 39:131-138). FIGS. 7a and 7b show the
fluorescence images of 4 optical sections of a Caco-2 cell
monolayer that had been incubated with the fCS-.gamma.-PGA
nanoparticles having a positive surface charge (0.10%
.gamma.-PGA:0.20% CS, zeta potential: about 21 mV) for 20 and 60
min, respectively. As shown, after 20 min of incubation with the
nanoparticles, intense fluorescence signals at intercellular spaces
were observed at depths of 0 and 5 .mu.m from the apical (upper)
surface of the cell monolayer. The intensity of fluorescence became
weaker at levels deeper than 10 .mu.m from the apical surface of
the cell monolayer and was almost absent at depths.gtoreq.15 .mu.m
(FIG. 7a).
[0100] After 60 minutes of incubation with the nanoparticles, the
intensity of the fluorescence observed at intercellular spaces was
stronger and appeared at a deeper level than those observed at 20
min after incubation. These observations correlated with our TEER
results, confirming that the nanoparticles with a positive surface
charge (CS dominated on the surface) were able to open the tight
junctions between Caco-2 cells and allow transport of the
nanoparticles by passive diffusion via the paracellular
pathways.
Example No. 6
In Vivo Study with Fluorescence-Labeled Nanoparticles
[0101] Fluorescence (FITC)-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA)
nanoparticles were prepared for the confocal laser scanning
microscopy (CLSM) study. After feeding rats with fCS-.gamma.-PGA
nanoparticles, the rats were sacrificed at a pre-determined time
and the intestine isolated for CLSM examination. The fluorescence
images of the nanoparticles that showed penetration through the
mouse intestine at appropriate time and at various depths from the
inner surface toward the exterior surface of the intestine,
including duodenum, jejunum, and ileum were clearly observed by
CLSM.
Example No. 7
Insulin Loading Capacity in Nanoparticles
[0102] Fluorescence (FITC)-labeled .gamma.-PGA was added into the
chitosan solution to prepare fluorescence (FITC)-labeled,
insulin-loaded CS-.gamma.-PGA nanoparticles for in vivo animal
study with confocal laser scanning microscopy (CLSM) assessment and
bioactivity analysis. The insulin-loaded CS-.gamma. PGA
nanoparticles are prepared by using the ionic-gelation method upon
addition of insulin mixed with .gamma.-PGA solution into CS
solution, followed by magnetic stirring in a container.
[0103] Model insulin used in the experiment and disclosed herein is
obtained from bovine pancreas (Sigma-Aldrich, St. Louis, Mo.),
having a molecular formula of
C.sub.254H.sub.377N.sub.65O.sub.75S.sub.6 with a molecular weight
of about 5733.5 and an activity of .gtoreq.27 USP units/mg. The
insulin contains a two-chain polypeptide hormone produced by the
.beta.-cells of pancreatic islets. The .alpha. and .beta. chains
are joined by two interchain disulfide bonds. Insulin regulates the
cellular uptake, utilization, and storage of glucose, amino acids,
and fatty acids as well as inhibits the breakdown of glycogen,
protein, and fat. The insulin from Sigma-Aldrich contains about
0.5% zinc. Separately, insulin can be obtained from other sources,
such as a human insulin solution that is chemically defined,
recombinant from Saccharomyces cerevisiae. Some aspects of the
invention relate to nanoparticles with insulin in the core, wherein
the insulin may contain intermediate-acting, regular insulin,
rapid-acting insulin, sustained-acting insulin that provides slower
onset and longer duration of activity than regular insulin, or
combinations thereof.
[0104] Examples of insulin or insulin analog products include, but
not limited to, Humulin.RTM. (by Eli Lilly), Humalog.RTM. (by Eli
Lilly) and Lantus.RTM. (by Aventis), and Novolog.RTM. Mix70/30 (by
Novo Nordisk). Humalog (insulin lispro, rDNA origin) is a human
insulin analog that is a rapid-acting, parenteral blood
glucose-lowering agent. Chemically, it is Lys(B28), Pro(B29) human
insulin analog, created when the amino acids at positions 28 and 29
on the insulin B-chain are reversed. Humalog is synthesized with a
special non-pathogenic laboratory strain of Escherichia coli
bacteria that has been genetically altered by the addition of the
gene for insulin lispro. Humalog has the empirical formula
C.sub.257H.sub.383N.sub.65O.sub.77S.sub.6 and a molecular weight of
5808, identical to that of human insulin. The vials and cartridges
contain a sterile solution of Humalog for use as an injection.
Humalog injection consists of zinc-insulin lispro crystals
dissolved in a clear aqueous fluid. Each milliliter of Humalog
injection contains insulin lispro 100 Units, 16 mg glycerin, 1.88
mg dibasic sodium phosphate, 3.15 mg m-cresol, zinc oxide content
adjusted to provide 0.0197 mg zinc ion, trace amounts of phenol,
and water for injection. Insulin lispro has a pH of 7.0-7.8.
Hydrochloric acid 10% and/or sodium hydroxide 10% may be added to
adjust pH.
[0105] Humulin is used by more than 4 million people with diabetes
around the world every day. Despite its name, this insulin does not
come from human beings. It is identical in chemical structure to
human insulin and is manufactured in a factory using a chemical
process called recombinant DNA technology. Humulin L is an
amorphous and crystalline suspension of human insulin with a slower
onset and a longer duration of activity (up to 24 hours) than
regular insulin. Humulin U is a crystalline suspension of human
insulin with zinc providing a slower onset and a longer and less
intense duration of activity (up to 28 hours) compared to regular
insulin or the intermediate-acting insulins (NPH and Lente).
[0106] LANTUS.RTM. (insulin glargine [rDNA origin] injection) is a
sterile solution of insulin glargine for use as an injection.
Insulin glargine is a recombinant human insulin analog that is a
long-acting (up to 24-hour duration of action), parenteral
blood-glucose-lowering agent. LANTUS is produced by recombinant DNA
technology utilizing a non-pathogenic laboratory strain of
Escherichia coli (K12) as the production organism. Insulin glargine
differs from human insulin in that the amino acid asparagine at
position A21 is replaced by glycine and two arginines are added to
the C-terminus of the B-chain. Chemically, it is
21.sup.A-Gly-30.sup.Ba-L-Arg-30.sup.Bb-L-Arg-human insulin and has
the empirical formula C.sub.267H.sub.404N.sub.72O.sub.78S.sub.6
with a molecular weight of 6063.
[0107] LANTUS consists of insulin glargine dissolved in a clear
aqueous fluid. Each milliliter of LANTUS (insulin glargine
injection) contains 100 IU (3.6378 mg) insulin glargine. Inactive
ingredients for the 10 mL vial are 30 mcg zinc, 2.7 mg m-cresol, 20
mg glycerol 85%, 20 mcg polysorbate 20, and water for injection.
Inactive ingredients for the 3 mL cartridge are 30 mcg zinc, 2.7 mg
m-cresol, 20 mg glycerol 85%, and water for injection. In 2006,
there were 11.4 million prescriptions of Lantus in the U.S. for
basal insulin maintenance.
[0108] Novolog.RTM. Mix70/30 (70% insulin aspart protamine
suspension and 30% insulin aspart injection [rDNA origin]) is a
human insulin analog suspension. Novolog.RTM. Mix70/30 is a blood
glucose-lowering agent with a rapid onset and an intermediate
duration of action. Insulin aspart is homologous with regular human
insulin with the exception of a single substitution of the amino
acid praline by aspartic acid in position B28, and is produced by
recombinant DNA technology utilizing Saccharomyces cerevisiae as
the production organism. Insulin aspart (Novolog) has the empirical
formula C.sub.256H.sub.381N.sub.65O.sub.79S.sub.6 and a molecular
weight of 5826. Novolog.RTM. Mix70/30 is a uniform, white sterile
suspension that contains zinc 19.6 .mu.g/ml and other
components.
[0109] The nanoparticles with two insulin concentrations are
prepared at a chitosan to .gamma.-PGA ratio of 0.75 mg/ml to 0.167
mg/ml. Their particle size and zeta potential are shown in Table 3
below.
TABLE-US-00004 TABLE 3 Insulin Conc. Mean Particle Polydispersity
Zeta Potential (mg/ml) (n = 5) Size (nm) Index (PI) (mV) 0* 145.6
.+-. 1.9 0.14 .+-. 0.01 +32.11 .+-. 1.61 0.042 185.1 .+-. 5.6 0.31
.+-. 0.05 +29.91 .+-. 1.02 0.083 198.4 .+-. 6.2 0.30 .+-. 0.09
+27.83 .+-. 1.22 *control reference without insulin
[0110] Further, their association efficiency of insulin and loading
capacity of insulin are analyzed, calculated and shown in FIGS. 11
and 12, according to the following formula:
Insulin Association = ( Total amount of insulin - Insulin in
supernatant ) Total amount of insulin .times. 100 % ##EQU00001##
Efficiency ( LE % ) Loading Capacity ( LC ) = ( Total amount of
insulin - Insulin in supernatant ) Weight of recovered particles
.times. 100 % ##EQU00001.2##
[0111] FIG. 11 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan and .gamma.-PGA, whereas FIG.
12 shows loading capacity and association efficiency of insulin in
nanoparticles of chitosan alone (in absence of .gamma.-PGA) as
reference. The data clearly demonstrates that both the insulin
loading capacity and insulin association efficiency are
statistically higher for the nanoparticles with .gamma.-PGA in the
core. The LE (40.about.55%) and LC (5.0.about.14.0%) of insulin for
CS-.gamma. PGA nanoparticles were obtained by using the
ionic-gelation method upon addition of insulin mixed with
.gamma.-PGA solution into CS solution, followed by magnetic
stirring for nanoparticle separation.
[0112] In certain follow-up experiments, nanoparticles having a
pharmaceutical composition have been successfully prepared with a
negatively charged component comprised of .gamma.-PGA, .alpha.-PGA,
PGA derivatives, salts of PGA, heparin or heparin analog,
glycosaminoglycans, or alginate. The PGA derivatives of the present
invention may include, but are not limited to,
poly-.gamma.-glutamic acid, poly-.alpha.-glutamic acid,
poly-L-glutamic acid (manufactured by Sigma-Aldrich, St. Louis,
Mo.), poly-D-glutamic acid, poly-L-.alpha.-glutamic acid,
poly-.gamma.-D-glutamic acid, poly-.gamma.-DL-glutamic acid, and
PEG or PHEG derivatives of polyglutamic acid, salts of the
above-cited PGAs, and the like. Some aspects of the invention
relate to nanoparticles comprising a shell component and a core
component, wherein at least a portion of the shell component
comprises chitosan and wherein the core component is comprised of a
negatively charged compound that is conjugated to chitosan, and a
bioactive agent. Some aspects of the invention relate to an oral
dose of nanoparticles that effectively enhance epithelial
permeation (such as intestinal or blood brain paracellular
transport) comprising a negative component (such as .gamma.-PGA,
.alpha.-PGA, PGA derivatives, heparin, or alginate) in the core and
low molecular weight chitosan, wherein the chitosan dominates on a
surface of the nanoparticles with positive charges.
[0113] Some aspects of the invention relate to a dose of
nanoparticles that effectively enhance epithelial permeation,
intestinal transport or blood brain paracellular transport
comprising a polyanionic component (such as .gamma.-PGA,
.alpha.-PGA, PGA derivatives, heparin, heparin analogs, low
molecular weight heparin, glycosaminoglycans, or alginate) in the
core and low molecular weight chitosan in the shell, wherein the
chitosan dominates on the surface of the nanoparticles with
positive surface charges. In practice, the Alzheimer's drug is
encapsulated in the chitosan shell nanoparticle as described
herein, wherein the nanoparticle is partially crosslinked
(optionally) to enhance its biodurability. Then, the nanoparticles
are intra-venously injected, whereby the nanoparticles pass to the
brain in blood circulation. The chitosan shell of the nanoparticles
adheres to the surface adjacent the tight junction in the brain.
Thereafter, the chitosan nanoparticle opens the tight junction,
wherein the Alzheimer's drug is released for therapeutic treatment
after passing the tight junction. In one embodiment, the
nanoparticles are in a spherical shape having a mean particle size
of about 50 to 250 nanometers, preferably 150 nanometers to 250
nanometers.
[0114] Dalteparin is a low molecular weight heparin. It is marketed
as Fragmin.RTM. by Pfizer Inc. Like other low molecular weight
heparins, dalteparin is used for prophylaxis or treatment of deep
vein thrombosis and pulmonary embolism. The CLOT study, published
in 2003, showed that in patients with malignancy and acute venous
thromboembolism, dalteparin was more effective than Coumadin in
reducing the risk of recurrent embolic events. Dalteparin is the
only low molecular weight heparin shown to be safe in critically
ill people with renal failure. Heparins are cleared by the kidneys,
but studies have shown that dalteparin does not accumulate even if
kidney function is reduced.
[0115] In one example, intravenous administration of the
nanoparticles comprising chitosan shell substrate, polyanionic core
substrate and at least one bioactive agent for treating Alzheimer's
disease in an animal subject is typically performed with 10 mg to
40 mg of active agent per day over a period of one month to one
year. The bioactive agent is selected from the group consisting of
donepezile, rivastignine, galantamine, and/or those trade-named
products, such as memantine hydrochloride (Axura.RTM. by Merz
Pharmaceuticals), donepezil hydrochloride (Aricept.RTM. by Eisai
Co. Ltd.), rivastigmine tartrate (Exelon.RTM. by Novartis),
galantamine hydrochloride (Reminyl.RTM. by Johnson & Johnson),
and tacrine hydrochloride (Cognex.RTM. by Parke Davis).
[0116] Some aspects of the invention relate to a nanoparticle with
a core substrate comprising polyglutamic acids such as water
soluble salt of polyglutamic acids (for example, ammonium salt) or
metal salts of polyglutamic acid (for example, lithium salt, sodium
salt, potassium salt, magnesium salt, and the like). In one
embodiment, the polyglutamic acid may be selected from the group
consisting of poly-.alpha.-glutamic acid, poly-L-.alpha.-glutamic
acid, poly-.gamma.-glutamic acid, poly-D-glutamic acid,
poly-.gamma.-D-glutamic acid, poly-.gamma.-DL-glutamic acid,
poly-L-glutamic acid (manufactured by Sigma-Aldrich, St. Louis,
Mo.), and PEG or PHEG derivatives of polyglutamic acid. Alginate is
generally non-biodegradable; however, it is stipulated that an
alginate particle with about 30-50 kDa molecular weight is kidney
inert. Heparin with negatively charged side-groups has a general
chemical structure as shown below:
##STR00002##
[0117] Some aspects of the invention relate to the negatively
charged glycosaminoglycans (GAGs) as the core substrate of the
present nanoparticles. GAGs may be complexed with a
low-molecular-weight chitosan to form drug-carrier nanoparticles.
GAGs may also conjugate with the protein drugs as disclosed herein
to enhance the bonding efficiency of the core substrate in the
nanoparticles. Particularly, the negatively charged core substrate
(such as GAGs, heparin, PGA, alginate, and the like) of the
nanoparticles of the present invention may conjugate with
chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF, MK, VEGF,
KGF, bFGF, aFGF, MK, PTN, etc.
[0118] Anti-Diabetic Drugs
[0119] The anti-diabetic drugs or drugs for treating diabetes are
broadly categorized herein as insulin/insulin analogs anti-diabetic
drugs and non-insulin anti-diabetic drugs. The non-insulin
anti-diabetic drugs may include, but not limited to, insulin
sensitizers, such as biguanides (for example, metformin, buformin,
phenoformin, and the like), thiazolidinedione (TZDs; for example,
pioglitazone, rivoglitazone, rosiglitazone, troglitazone, and the
like), and dual PPAR agonists (for example aleglitazar,
muraglitazar, tesaglitazar, and the like; PPAR is abbreviation for
peroxisome proliferator-activated receptor). The non-insulin
anti-diabetic drugs may also include, but not limited to,
secretagogues, such as sulfonylureas (for example, carbutamide,
chlopropamide, gliclazide, tolbutamide, tolazamide, glipizide,
glibenclamide, gliquidone, glyclopyramide, glimepiride, and the
like), meglitinides (for example, nateglinide, repaglinide,
mitiglinide, and the like), GLP-1 analogs (for example, exenatide,
liraglutide, albiglutide, lixisenatide, taspoglutide, and the
like), and DPP-4 inhibitors (for example, alogliptin, linagliptin,
saxagliptin, sitagliptin, vildagliptin, and the like; DPP-4 is
abbreviation for inhibitor of dipeptidyl peptidase 4). Further, the
non-insulin anti-diabetic drugs may include, but not limited to,
alpha-glucosidase inhibitors (for example, acarbose, miglitol,
voglibose, and the like), amylin analog (for example, pramlintide
and the like), SGLT2 inhibitor (for example, dapagliflozin,
remogliflozin, sergliflozin, and the like), benfluorex, and
tolrestat. Here, The sodium-glucose co-transporter type 2 (SGLT2)
is a 672 amino acid high-capacity low-affinity transporter
expressed in the S1 segment of the proximal tubule which is
believed to mediate the majority of renal glucose reabsorption.
[0120] In one embodiment, the anti-diabetic drug may comprise
osteocalcin. Osteocalcin, also known as bone gamma-carboxyglutamic
acid-containing protein (BGLAP), is a noncollagenous protein found
in bone and dentin. Osteocalcin acts as a hormone in the body,
causing beta cells in the pancreas to release more insulin, and at
the same time directing fat cells to release the hormone
adiponectin, which increases sensitivity to insulin.
[0121] Glucagon, a hormone secreted by the pancreas, raises blood
glucose levels. Its effect is opposite that of insulin, which
lowers blood glucose levels. The pancreas releases glucagon when
blood sugar (glucose) levels fall too low. Glucagon causes the
liver to convert stored glycogen into glucose, which is released
into the bloodstream. Glucagon also stimulates the release of
insulin, so glucose can be taken up and used by insulin-dependent
tissues. Thus, glucagon and insulin are part of a feedback system
that keeps blood glucose levels at a stable level. Glucagon belongs
to a family of several other related hormones. In one embodiment,
the first bioactive agent (for example, glucagon) of the present
invention is compatible with the second bioactive agent (for
example, insulin), wherein glucagon stimulates the release of
insulin, so glucose can be taken up and used by insulin-dependent
tissues, when both are co-administered to an animal subject. Thus,
glucagon and insulin are part of a feedback system that keeps blood
glucose levels at a stable level.
[0122] Clinical data have shown enhanced anti-diabetic efficiency
(or glycemic control) in an animal subject by co-administering two
different anti-diabetic drugs; for example, exenatide has been
approved to be co-administered along with metformin, or a
combination of metformin and a sulfonyurea, or thiazolidinediones
(such as pioglitazone or rosiglitazone). In cholesterol management
clinical trials, it was reported that the combination of
bezafibrate and diffunisol produced better clinical data than
bezafibrate alone. Bezafibrate (marketed as Bezalip and various
other brand names) is a fibrate drug used for the treatment of
hyperlipidaemia. It helps to lower LDL cholesterol and triglyceride
in the blood, and increase HDL.
[0123] Some aspects of the invention relate to a therapeutic method
of treating a subject by co-administering at least two nanoparticle
compositions, the first nanoparticle composition of the present
invention comprising a first bioactive agent, wherein the second
nanoparticle composition of the present invention comprises a
second bioactive agent that is different from the first bioactive
agent. In one example, the first bioactive agent is exenatide in
the first nanoparticles and the second bioactive agent is metformin
in the second nanoparticles. In one embodiment, the delivery route
of administering the first nanoparticle composition is different
from the delivery route of administering the second of the at least
two nanoparticle compositions. In one embodiment, both types of
first and second nanoparticles are loaded in the same capsules. In
another embodiment, the first nanoparticles and the second
nanoparticles are loaded in separate different capsules. In a
further embodiment, the first bioactive agent is compatible with
the second bioactive agent and optionally, the first bioactive
agent enhances the therapeutic effects of the second bioactive
agent when they are co-administered to the subject.
[0124] Some aspects of the invention relate to a system of
pharmaceutical composition comprising two distinct types of
bioactive nanoparticles, wherein the first type of bioactive
nanoparticles comprises a shell portion that is dominated by
positively charged chitosan, a core portion that contains
negatively charged substrate, wherein the negatively charged
substrate is at least partially neutralized with a portion of the
positively charged chitosan and at least a first bioactive agent,
and wherein the second type of bioactive nanoparticles comprises a
shell portion that is dominated by positively charged chitosan, a
core portion that contains negatively charged substrate, wherein
the negatively charged substrate is at least partially neutralized
with a portion of the positively charged chitosan and at least a
second bioactive agent. In one embodiment, both types of the first
and second nanoparticles are loaded in the same capsules for
administering to a subject. In another embodiment, the first type
of nanoparticles and the second type of nanoparticles are loaded in
separate different capsules in the system for co-administering to a
subject.
[0125] Calceti et al. reported an in vivo evaluation of an oral
insulin-PEG delivery system (Eur J Pharma Sci 2004; 22:315-323).
Insulin-PEG was formulated into mucoadhesive tablets constituted by
the thiolated polymer poly(acrylic acid)-cysteine. The therapeutic
agent was released from these tablets within 5 hours in a sustained
manner. In vivo, by oral administration to diabetic mice, the
glucose levels were found to decrease significantly over the time.
Further, Krauland et al. reported another oral insulin delivery
study of thiolated chitosan-insulin tablets on non-diabetic rats
(J. Control. Release 2004, 95:547-555). The delivery tablets
utilized 2-Iminothiolane covalently linked to chitosan to form
chitosan-TBA (chitosan-4-thiobutylamidine) conjugate. After oral
administration of chitosan-TBA-insulin tablets to non-diabetic
conscious rats, the blood glucose level decreased significantly for
24 hours; supporting the expected sustained insulin release of the
presently disclosed nanoparticles herein through intestinal
absorption. In a further report by Morcol et al. (Int. J. Pharm.
2004; 277:91-97), an oral delivery system comprising calcium
phosphate-PEG-insulin-casein particles displays a prolonged
hypoglycemic effect after oral administration to diabetic rats.
[0126] Pan et al. disclosed that chitosan nanoparticles improving
the intestinal absorption of insulin in vivo (Int J Pharma 2002;
249:139-147) with insulin-chitosan nanoparticles at a particle size
of 250-400 nm, a polydispersity index smaller than 0.1, positively
charged and stable. After administering the insulin-chitosan
nanoparticles, it was found that the hypoglycemic effect was
prolonged with enhanced pharmacological bioavailability. Their data
confirmed our observation as shown in FIGS. 11 and 12; however, the
insulin loading capacity and insulin association efficiency of the
present invention are substantially higher for the chitosan-insulin
nanoparticles with .gamma.-PGA in the core as the core
substrate.
Example No. 8
Insulin Nanoparticle Stability
[0127] FIG. 13 shows the stability of insulin-loaded nanoparticles
of the present invention with an exemplary composition of CS 0.75
mg/ml, .gamma.-PGA 0.167 mg/ml, and insulin 0.083 mg/ml. The
prepared insulin-loaded nanoparticles suspended in deionized water
are stable during storage up to 40 days. First (in FIG. 13), the
insulin content in the nanoparticle storage solution maintains at
about a constant level of 9.5%. The nanoparticle stability is
further evidenced by the substantially constant particle size at
about 200 nm and substantially constant zeta potential of about +28
mV over the period of about 40 days. It is contemplated that the
insulin-containing nanoparticles of the present invention would
further maintain their biostability when formulated in a soft
gelcap or capsule configuration that further isolates the
nanoparticles from environmental effects, such as sunlight, heat,
air conditions, and the like. Some aspects of the invention provide
a gelcap pill or capsule containing a dosage of insulin
nanoparticles effective amount of the insulin to treat or manage
the diabetic animal subjects, wherein the stability of the
insulin-containing nanoparticles is at least 40 days, preferably
more than 6 months, and most preferably more than a couple of
years.
[0128] By "effective amount of the insulin", it is meant that a
sufficient amount of insulin will be present in the dose to provide
for a desired therapeutic, prophylatic, or other biological effect
when the compositions are administered to a host in single dosage
forms. The capsule of the present invention may preferably comprise
two-part telescoping gelatin capsules. Basically, the capsules are
made in two parts by dipping metal rods in molten gelatin solution.
The capsules are supplied as closed units to the pharmaceutical
manufacturer. Before use, the two halves are separated, the capsule
is filled with powder (either by placing a compressed slug of
powder into one half of the capsule, or by filling one half of the
capsule with loose powder) and the other half of the capsule is
pressed on. The advantage of inserting a slug of compressed powder
is that there is a superior control of weight variation. The
capsules may be enterically coated before filling the powder or
after securing both parts of the filled capsule together. In one
embodiment, the capsules further comprise a permeation enhancer,
wherein the permeation enhancer is selected from the group
consisting of Ca.sup.2+ chelators, bile salts, anionic surfactants,
medium-chain fatty acids, phosphate esters, chitosan, and chitosan
derivatives.
[0129] In another embodiment, the capsule may contain solubilizer,
bubbling agent, emulsifier, or other pharmacopoeial excipients,
such as Generally Recognized as Safe (GRAS). GRAS is a United
States of America Food and Drug Administration (FDA) designation
that a chemical or substance added to food is considered safe by
experts, and therefore exempted from the usual Federal Food, Drug,
and Cosmetic Act (FFDCA) food additive tolerance requirements. The
bubbling agent is the agent that emits carbon dioxide gas when
contacting liquid with a purpose to burst the capsule or promote
intimate contact of the capsule content with the surrounding
material outside of the capsule. For example, reaction of sodium
bicarbonate and an acid to give a salt and carbonic acid, which
readily decomposes to carbon dioxide and water. The bubbling agent
may include sodium bicarbonate/citric acid mixture, Ac-Di-Sol, and
the like. The chemical Ac-Di-Sol has the IUPAC name of acetic acid,
2,3,4,5,6-pentahydroxyhexanal, sodium and a chemical formula of
C.sub.8H.sub.16NaO.sub.8. An emulsifier is a substance that
stabilizes an emulsion by increasing its kinetic stability. One
class of emulsifiers is known as surface active substances, or
surfactants. Detergents are another class of surfactant emulsifier,
and will physically interact with both oil and water, thus
stabilizing the interface between oil or water droplets in
suspension. The most popular emulsions are non-ionic because they
have low toxicity. Cationic emulsions may also be used herein
because their antimicrobial properties.
[0130] Thus, for convenient and effective oral administration,
pharmaceutically effective amounts of the nanoparticles of this
invention can be tabletted with one or more excipient, encased in
capsules such as gel capsules, or suspended in a liquid solution
and the like. The nanoparticles can be suspended in a deionized
solution or a similar solution for parenteral administration. The
nanoparticles may be formed into a packed mass for ingestion by
conventional techniques. For instance, the nanoparticles may be
encapsulated as a "hard-filled capsule" or a "soft-elastic capsule"
using known encapsulating procedures and materials. The
encapsulating material should be highly soluble in gastric fluid so
that the particles would be rapidly dispersed in the stomach after
the capsule is ingested. Each unit dose, whether capsule or tablet,
will preferably contain nanoparticles of a suitable size and
quantity that provides pharmaceutically effective amounts of the
nanoparticles. The applicable shapes and sizes of capsules may
include round, oval, oblong, tube or suppository shape with sizes
from 0.75 mm to 80 mm or larger. The volume of the capsules can be
from 0.05 cc to more than 5 cc. In one embodiment, the interior of
capsules is treated to be hydrophobic or lipophilic.
Example No. 9
In Vitro Insulin Release Study
[0131] FIG. 14 show a representative protein drug (for example,
insulin) release profile in a pH-adjusted solution for
pH-sensitivity study with an exemplary composition of CS 0.75
mg/ml, .gamma.-PGA 0.167 mg/ml, and insulin 0.083 mg/ml in
nanoparticles. In one embodiment, the exemplary composition may
include each component at a concentration range of +10% as follows:
CS 0.75 mg/ml (a concentration range of 0.67 to 0.83 mg/ml),
.gamma.-PGA 0.167 mg/ml (a concentration range of 0.150 to 0.184
mg/ml), and insulin 0.083 mg/ml (a concentration range of 0.075 to
0.091 mg/ml). First, solution of the insulin-loaded nanoparticles
was adjusted to pH 2.5 to simulate the gastric environment in a
DISTEK-2230A container at 37.degree. C. and 100 rpm. Samples (n=5)
were taken at a pre-determined particular time interval and the
particle-free solution was obtained by centrifuging at 22,000 rpm
for 30 minutes to analyze the free or released insulin in solution
by HPLC. Until the free insulin content in the sample solution
approaches about constant of 26% (shown in FIG. 14), the pH was
adjusted to 6.6 to simulate the entrance portion of the intestine.
The net released insulin during this particular time interval is
about (from 26% to 33%) 7%. In other words, the nanoparticles are
quite stable (evidenced by minimal measurable insulin in solution)
for both the pH 2.5 and pH 6.6 regions.
[0132] To simulate the exit portion of the intestine, the
insulin-containing nanoparticle solution was adjusted to pH 7.4.
The remaining insulin (about 67%) was released from the
nanoparticles at this time. As discussed above, the insulin in
nanoparticles would be more effective to penetrate the intestine
wall in paracellular transport mode than the free insulin because
of the present invention of the nanoparticles with chitosan at the
outer surface (preferential mucosal adhesion on the intestinal
wall) and positive charge (enhancing paracellular tight junction
transport).
[0133] Since chitosan-shelled nanoparticles exhibit positive
surface charge and preferential mucoadhesive properties (both are
required for enhancing paracellular permeation), some aspects of
the invention relate to a method of coating a nanoparticle (for
example, a nanoparticle with little or no chitosan) with chitosan
solution, resulting in a chitosan-shelled nanoparticle with
positive surface charge.
Example No. 10
In Vivo Study with Insulin-Loaded Fluorescence-Labeled
Nanoparticles
[0134] In the in vivo study, rats were injected with streptozotocin
(STZ 75 mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to
induce diabetes. The blood from the rat's tail was analyzed with a
commercially available glucometer for blood glucose. The blood
glucose level on Wistar male rats at no fasting (n=5) was measured
at 107.2.+-.8.1 mg/dL for normal rats while the blood glucose level
was at 469.7.+-.34.2 mg/dL for diabetic rats. In the animal study,
diabetic rats were fasting for 12 hours and subjected to four
different conditions: (a) oral deionized water (DI) administration;
(h) oral insulin administration at 30 U/kg; (c) oral insulin-loaded
nanoparticles administration at 30 U/kg; and (d) subcutaneous (SC)
insulin injection at 5 U/kg as positive control. The blood glucose
concentration from rat's tail was measured over the time in the
study.
[0135] FIG. 15 shows glucose change (hypoglycemic index) versus
time of the in vivo animal study (n=5). The glucose change as a
percentage of base lines (the base line was the glucose level in an
animal subject without the effect of insulin) for both oral DI
administration and oral insulin administration over a time interval
of 8 hours appears relatively constant within the experimental
measurement error range. It is illustrative that substantially all
insulin from the oral administration route has been decomposed in
rat stomach. As anticipated, the glucose decrease for the SC
insulin injection route appears in rat blood in the very early time
interval and starts to taper off after 3 hours in this exemplary
study.
[0136] The most important observation of the study comes from the
oral administration route with insulin-loaded nanoparticles. The
blood glucose begins to decrease from the base line at about 2
hours after administration and sustains a lower glucose level at
more than 8 hours into study. It implies that the current
insulin-loaded nanoparticles may modulate the glucose level in
animals in a sustained or prolonged effective mode. Some aspects of
the invention provide a method of treating diabetes of an animal
subject comprising orally administering insulin-containing
nanoparticles with a dosage effective amount of the insulin to
treat the diabetes, wherein at least a portion of the nanoparticles
comprises a positively charged shell substrate and a negatively
charged core substrate. In one embodiment, the dosage effective
amount of the insulin to treat the diabetes comprises an insulin
amount of between about 15 units to 45 units per kilogram body
weight of the animal subject, preferably 20 to 40 units, and most
preferably at about 25 to 35 units insulin per kilogram body
weight.
[0137] Some aspects of the invention relate to a novel nanoparticle
system that is composed of a low-MW CS and .gamma.-PGA with CS
dominated on the surfaces being configured to effectively open the
tight junctions between Caco-2 cell monolayers. The surface of the
nanoparticles is characterized with a positive surface charge. In
one embodiment, the nanoparticles of the invention enables
effective intestinal delivery for bioactive agent, including
peptide, polypeptide, protein drugs, other large hydrophilic
molecules, and the like. Such polypeptide drugs can be any natural
or synthetic polypeptide that may be orally administered to a
patient or an animal subject.
[0138] Exemplary drugs include, but are not limited to, insulin;
growth factors, such as epidermal growth factor (EGF), insulin-like
growth factor (IGF), transforming growth factor (TGF), nerve growth
factor (NGF), platelet-derived growth factor (PDGF), bone
morphogenic protein (BMP), fibroblast growth factor and the like;
hemophilia factors, somatostatin; somatotropin; somatropin;
somatrem; calcitonin; parathyroid hormone; colony stimulating
factors (CSF); clotting factors; tumor necrosis factors:
interferons; interleukins; gastrointestinal peptides, such as
vasoactive intestinal peptide (VIP), cholecytokinin (CCK), gastrin,
secretin, and the like; erythropoietins; growth hormone and GRF;
vasopressins; octreotide; pancreatic enzymes; dismutases such as
superoxide dismutase; thyrotropin releasing hormone (TRH); thyroid
stimulating hormone; luteinizing hormone; LHRH; GHRH; tissue
plasminogen activators; macrophage activator; chorionic
gonadotropin; heparin; atrial natriuretic peptide; hemoglobin;
retroviral vectors; relaxin; cyclosporin; oxytocin; vaccines;
monoclonal antibodies; and the like; and analogs and derivatives of
these compounds.
[0139] Triptorelin (acetate or pamoate), a decapeptide
(pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), is a
gonadotropin-releasing hormone agonist (GnRH agonist). By causing
constant stimulation of the pituitary, it decreases pituitary
secretion of gonadotropins luteinizing hormone (LH) and follicle
stimulating hormone (FSH). Like other GnRH agonists, triptorelin
may be used in the treatment of hormone-responsive cancers such as
prostate cancer or breast cancer, precocious puberty,
estrogen-dependent conditions (such as endometriosis or uterine
fibroids), and in assisted reproduction. Triptorelin is marketed
under the brand names Decapeptyl (Ipsen) and Diphereline and
Gonapeptyl (Ferring Pharmaceuticals). In the United States, it is
sold by Pfizer as Trelstar. Its systematic (IUPAC) name is
5-oxo-D-prolyl-L-histidyl-Ltryptophyl-L-seryl-Ltyrosyl-3-(1H-indol-2-yl)--
L-alanylleucyl-L-arginyl-L-prolylglycinamide. It has a chemical
formula C.sub.64H.sub.82N.sub.18O.sub.13 with a molecular mass
1311.5 g/mol.
[0140] Gemtuzumab ozogamicin (marketed by Wyeth as Mylotarg.RTM.)
is a monoclonal antibody used to treat acute myelogenous leukemia.
It is a monoclonal antibody to CD33 linked to a cytotoxic agent,
calicheamicin. CD33 is expressed in most leukemic blast cells but
also in normal hematopoietic cells, the intensity diminishing with
maturation of stem cells. The gemtuzumab ozogamicin has been
evaluated in the nanoparticle formulation of the present invention,
showing typical characteristics in a spheroidal configuration with
a particle size of between about 50 to 400 nm, a positive surface
charge, and a narrow polydispersity index.
[0141] The bioactive agent of the present invention may also be
selected from group consisting of oxytocin, vasopressin,
adrenocorticotrophic hormone, prolactin, luliberin or luteinising
hormone releasing hormone, growth hormone, growth hormone releasing
factor, somatostatin, glucagon, interferon, gastrin, tetragastrin,
pentagastrin, urogastroine, secretin, calcitonin, enkephalins,
endorphins, angiotensins, renin, bradykinin, bacitracins,
polymixins, colistins, tyrocidin, gramicidines, and synthetic
analogues, modifications and pharmacologically active fragments
thereof, monoclonal antibodies and soluble vaccines. Growth hormone
(GH) is a peptide hormone that stimulates growth and cell
reproduction in humans and other animals. It is a 191-amino acid,
single chain polypeptide hormone that is synthesized, stored, and
secreted by the somatotroph cells within the lateral wings of the
anterior pituitary gland. Somatotrophin refers to the growth
hormone produced natively in animals, the term somatropin refers to
growth hormone produced by recombinant DNA technology,.sup.[1] and
is abbreviated "rhGH" in humans.
[0142] In another embodiment, the nanoparticles of the invention
increase the absorption of bioactive agents across the blood brain
barrier and/or the gastrointestinal barrier. In still another
embodiment, the nanoparticles with chitosan dominant at an outer
layer that show positive surface charge serve as an enhancer in
enhancing drug (bioactive agent) permeation of an administered
bioactive agent when the bioactive agent and nanoparticles are
orally administrated in a two-component system, or orally
administered substantially simultaneously.
Example No. 11
Epithelial Permeation and Enhancers
[0143] Chitosan and its derivatives may function as epithelial
absorption enhancers. Chitosan, when protonated at an acidic pH, is
able to increase the permeability of peptide drugs across mucosal
epithelia. Some aspects of the invention provide co-administration
of nanoparticles of the present invention and at least one
permeation enhancer (in non-nanoparticle form or nanoparticle
form). In one embodiment, the nanoparticles can be formulated by
co-encapsulation of at least one permeation enhancer and at least
one bioactive agent, with an option of adding other components. In
one embodiment, the nanoparticles further comprise a permeation
enhancer. The permeation enhancer may be selected from the group
consisting of Ca.sup.2+ chelators, bile salts, anionic surfactants,
medium-chain fatty acids, phosphate esters, and chitosan or
chitosan derivatives. In one embodiment, the nanoparticles of the
present invention comprises a positively charged shell substrate
and a negatively charged core substrate, for example, nanoparticles
composed of .gamma.-PGA and chitosan that is characterized with a
substantially positive surface charge.
[0144] In some embodiments, the nanoparticles of the present
invention, or with at least one permeation enhancer are loaded in a
soft gel, pill, tablet, chewable, or capsule, or loaded in the
enteric coated counterpart of the soft gel, pill, tablet, chewable,
or capsule. The enhancers and the nanoparticles would arrive at the
tight junction about the same time to enhance transiently opening
the tight junction. In another embodiment, the at least one
permeation enhancer is co-enclosed within the shell of the
nanoparticles of the present invention. Therefore, some broken
nanoparticles or fragments would release enhancers to assist the
nanoparticles to open the tight junctions of the epithelial layers.
In an alternate embodiment, the at least one enhancer is enclosed
within a second nanoparticle having positive surface charges,
particularly a chitosan-type nanoparticle, wherein the second
nanoparticle is formulated without any bioactive agent or with a
different bioactive agent from that bioactive agent in the first
nanoparticle. When the drug-containing first nanoparticles of the
present invention are co-administered with the above-identified
second nanoparticles orally, the enhancers within the second
nanoparticles are released in the gastrointestinal tract to assist
the drug-containing first nanoparticles to open and pass the tight
junction or facilitate enhanced drug absorption and transport.
Example No. 12
Nanoparticles Loaded with Exenatide
[0145] Exenatide is a member of the class of drugs known as
incretin mimetics. Exenatide and pramlintide belong to a group of
non-insulin injectables for treatment of diabetes. Exenatide has a
molecular formula of C.sub.184H.sub.282N.sub.50O.sub.60S with a
molecular mass of about 4186.6 g/mol and an CAS no. 141732-76-5.
Exenatide is suitable to be incorporated in the core portion of
chitosan-shelled nanoparticles, wherein the core portion may
include positively charged chitosan and negatively charged core
substrate, such as .gamma.-PGA or .alpha.-PGA, with the option of
additional TPP and MgSO.sub.4 in the core portion. In preparation,
nanoparticles were obtained upon addition of a mixture of
.gamma.-PGA plus exenatide aqueous solution (pH 7.4, 2 ml), using a
pipette during the addition stage (0.5-5 ml, PLASTIBRAND.RTM.,
BrandTech Scientific Inc., Germany), into a low-MW CS aqueous
solution (pH 6.0, 10 ml) at concentrations higher than 0.10% by w/v
under magnetic stirring at room temperature to ensure positive
surface charge. Nanoparticles were collected by ultracentrifugation
at 38,000 rpm for 1 hour. Exenatide is wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water as
the solution products. The nanoparticles thus obtained via the
simple and mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge and a
narrow polydispersity index. In one embodiment, it may further be
encapsulated in capsules. In one embodiment, the interior surface
of the capsule is treated to be lipophilic or hydrophobic. In
another embodiment, the exterior surface of the capsule is
enteric-coated or treated with an enteric coating polymer. In a
preferred embodiment, the nanoparticles are further freeze-dried,
optionally being mixed with trehalose or with
hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.
[0146] Glucagon-like peptide-1 (GLP-1) is derived from the
transcription product of the proglucagon gene. The major source of
GLP-1 in the body is the intestinal L cell that secretes GLP-1 as a
gut hormone. The biologically active forms of GLP-1 are
GLP-1-(7-37) and GLP-1-(7-36)NH2. GLP-1 secretion by L cells is
dependent on the presence of nutrients in the lumen of the small
intestine. The secretagogues (agents that cause or stimulate
secretion) of this hormone include major nutrients like
carbohydrate, protein and lipid. Once in the circulation, GLP-1 has
a half-life of less than 2 minutes, due to rapid degradation by the
enzyme dipeptidyl peptidase-4 (DPP-4). Commercial GLP-1 ELISA kits
are generally available for GLP-1 assay.
[0147] Exenatide (marketed as Byetta) is the first of a new class
of medications (incretin mimetics) approved for the treatment of
type 2 diabetes. It is manufactured and marketed by Amylin
Pharmaceuticals and Eli Lilly and Company. Exenatide is a synthetic
version of exendin-4, a hormone in the saliva of the Gila monster,
a lizard native to several Southwestern American states. It
displays properties similar to human GLP-1. Exenatide is a
39-amino-acid peptide that mimics the GLP-1 incretin, an insulin
secretagogue with glucoregulatory effects. While it may lower blood
glucose levels on its own, it can also be combined with other
medications such as pioglitazone, metformin, sulfonylureas, and/or
insulin (not FDA approved yet) to improve glucose control. The
approved use of exenatide is with sulfonylureas, metformin or
thiazolinediones. The medication is injected subcutaneously twice
per day using a pre-filled pen device.
[0148] Typical human responses to exenatide include improvements in
the initial rapid release of endogenous insulin, suppression of
pancreatic glucagon release, delayed gastric emptying, and reduced
appetite--all of which function to lower blood glucose. Whereas
some other classes of diabetes drugs such as sulfonylureas,
thiazolinediones, and insulin are often associated with weight
gain, Byetta often is associated with significant weight loss.
Unlike sulfonylureas and meglitinides, exenatide only increases
insulin synthesis and secretion in the presence of glucose,
lessening the risk of hypoglycemia. Byetta is also being used by
some physicians to treat insulin resistance.
Example No. 13
Nanoparticles Loaded with Pramlintide
[0149] Pramlintide is a synthetic amylin analogue (marketed as
Symlin). Amylin is a natural, pancreatic islet peptide that is
normally secreted with insulin in response to meals. It has several
beneficial effects on glucose homeostasis: suppression of glucagon
secretion, delaying of gastric emptying, and promotion of satiety.
It is currently given before meals, in a separate subcutaneous
injection but usually in conjunction with insulin. Pramlintide has
a molecular formula of C.sub.171H.sub.169N.sub.51O.sub.53S.sub.2
with a molecular mass of about 3951.4 g/mol and an CAS no.
151126-32-8. Pramlintide (positively charged) is currently
delivered as an acetate salt. Pramlintide is suitable to be
incorporated in a core portion of chitosan-shelled nanoparticles,
wherein the core portion may include positively charged chitosan
and negatively charged core substrate, such as .gamma.-PGA or
.alpha.-PGA, with an option for additional TPP and MgSO.sub.4 in
the core portion. In other words, pramlintide may replace at least
a portion of positively charged chitosan in the core by interacting
with the negatively core substrate, such as PGA, heparin or the
like. In preparation, nanoparticles were obtained upon the addition
of a mixture of .gamma.-PGA plus pramlintide aqueous solution (pH
7.4, 2 ml), using a pipette during the addition step (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrations higher
than 0.10% by w/v under magnetic stirring at room temperature to
ensure positive surface charge.
[0150] Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour. Pramlintide is wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water as
the solution products. The nanoparticles thus obtained via the
simple and mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge and a
narrow polydispersity index. In one embodiment, it may further be
encapsulated in capsules. In one embodiment, the interior surface
of the capsule is treated to be lipophilic or hydrophobic. In
another embodiment, the exterior surface of the capsule is
enteric-coated. In a preferred embodiment, the nanoparticles are
further freeze-dried, and can be optionally mixed with trehalose or
with hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.
[0151] Pramlintide is an analogue of amylin, a small peptide
hormone that is released into the bloodstream after a meal by the
.beta.-cells of the pancreas along with insulin. Like insulin,
amylin is deficient in individuals with diabetes. By augmenting
endogenous amylin, pramlintide aids in the absorption of glucose by
slowing gastric emptying, promoting satiety via hypothalamic
receptors (different receptors than GLP-1), and inhibiting
inappropriate secretion of glucagon, a catabolic hormone that
opposes the effects of insulin and amylin.
Example No. 14
Nanoparticles Loaded with Complexed Calcitonin
[0152] Calcitonin is a protein drug that therapeutically serves as
calcium regulators for treating osteoporosis (J. Pharm. Pharmacol.
1994; 46:547-552). Calcitonin has a molecular formula of
C.sub.145H.sub.240N.sub.44O.sub.48S.sub.2 with a molecular weight
of about 3431.9 and an isoelectric point of 8.7. The net charge for
calcitonin at pH7.4 is positive that makes it suitable for
complexing or conjugating with negatively charged core substrate,
such as .gamma.-PGA or .alpha.-PGA. In preparation, nanoparticles
were obtained upon the addition of a mixture of .gamma.-PGA and
calcitonin aqueous solution (pH 7.4, 2 ml), using a pipette during
the addition step (0.5-5 ml, PLASTIBRAND.RTM., BrandTech Scientific
Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml)
at concentrations higher than 0.10% by w/v under magnetic stirring
at room temperature to ensure positive surface charge.
Nanoparticles were collected by ultracentrifugation at 38,000 rpm
for 1 hour. Calcitonin is wholly or substantially totally
encapsulated within the nanoparticles. Supernatants were discarded
and nanoparticles were resuspended in deionized water as the
solution products, further encapsulated in capsules or further
treated with an enteric coating polymer. The nanoparticles thus
obtained via the simple and mild ionic-gelation method described
herein show typical characteristics in a spheroidal configuration
with a particle size of between about 50 to 400 nm, a positive
surface charge and a narrow polydispersity index.
Example No. 15
Nanoparticles Loaded with Teriparatide
[0153] Teriparatide (Forteo.RTM.) is a recombinant form of
parathyroid hormone, used in the treatment of some forms of
osteoporosis. It is manufactured and marketed by Eli Lilly and
Company. Currently teriparatide is administered by injection once a
day in the thigh or abdomen. The recommended dose is 20 .mu.g per
day. Teriparatide has the chemical formula
C.sub.181H.sub.291N.sub.55O.sub.51S.sub.2 with a molecular mass of
4117.72 g/mol. Teriparatide is the portion of human parathyroid
hormone (PTH), amino acid sequence 1 through 34 of the complete
molecule which contains amino acid sequence 1 to 84. Endogenous PTH
is the primary regulator of calcium and phosphate metabolism in
bone and kidneys. Daily injections of teriparatide stimulate new
bone formation leading to increased bone mineral density.
[0154] Teriparatide is the first FDA approved agent for the
treatment of osteoporosis that stimulates new bone formation. In
one exemplary preparation, nanoparticles were obtained upon
addition of a mixture of .gamma.-PGA plus teriparatide aqueous
solution (2 ml), using a pipette (0.5-5 ml, PLASTIBRAND.RTM.,
BrandTech Scientific Inc., Germany) during the addition step, into
a low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrations
higher than 0.10% by w/v under magnetic stirring at room
temperature to ensure positive surface charge. Nanoparticles were
collected by ultracentrifugation at 38,000 rpm for 1 hour.
Teriparatide is wholly or substantially totally encapsulated within
the nanoparticles. Supernatants were discarded and nanoparticles
were resuspended in deionized water as the solution products,
further encapsulated in capsules/enteric capsules or further
treated with lyophilization. The nanoparticles thus obtained via
the simple and mild ionic-gelation method described herein show
typical characteristics in a spheroidal configuration with a
particle size of between about 50 to 400 nm, a positive surface
charge and a narrow polydispersity index.
Example No. 16
Nanoparticles Loaded with Vancomycin
[0155] Vancomycin is a protein drug that serves therapeutically as
an antibiotic against bacterial pathogens. Vancomycin has a
molecular formula of C.sub.66H.sub.75N.sub.9O.sub.24 with a
molecular weight of about 1485.7 and an isoelectric point of 5.0.
The net charge for vancomycin at pH7.4 is negative, which is
suitable to complex or conjugate with a portion of negatively
charged shell substrate, such as chitosan. In preparation,
nanoparticles were obtained upon addition of a mixture of
.gamma.-PGA and vancomycin aqueous solution (pH 7.4, 2 ml), using a
pipette (0.5-5 ml, PLASTIBRAND.RTM., BrandTech Scientific Inc.,
Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) with
excess concentrations under magnetic stirring at room temperature,
wherein CS concentration is provided sufficiently to conjugate
vancomycin, to counterbalance .gamma.-PGA, and exhibit positive
surface charge for the nanoparticles. Nanoparticles were collected
by ultracentrifugation at 38,000 rpm for 1 hour. Vancomycin is
wholly or substantially totally encapsulated within the
nanoparticles. Supernatants were discarded and nanoparticles were
resuspended in deionized water as the solution products, further
encapsulated in capsules or further treated with an enteric coating
on capsules. The nanoparticles thus obtained via the simple and
mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge and a
narrow polydispersity index.
[0156] Tigecycline is an glycylcycline antibiotic developed and
marketed by Wyeth under the brand name Tygacil.RTM.. It was
developed in response to the growing prevalence of antibiotic
resistance in bacteria such as Staphylococcus aureus. It has a
systematic (IUPAC) name
N-[(5aR,6aS,7S,9Z,10aS)-9-[amino(hydroxy)methylidene]-4,7-bis(dimethylami-
no)-1,10a,12-trihydroxy-8,10,11-trioxo-5,5a,6,6a,7,8,9,10,10a,11-decahydro-
tetracen-2-yl]-2-(tert-butylamino)acetamide. The chemical formula
is C.sub.29H.sub.39N.sub.5O.sub.8 with a molecular mass 585.65
g/mol. The drug inhibits the bacterial 30S ribosome and is
bacteriostatic. Tigecycline is active against many Gram-positive
bacteria, Gram-negative bacteria and anaerobes--including activity
against methicillin-resistant Staphylococcus aureus (MRSA) and
multi-drug resistant strains of Acinetobacter baumannii.
Tigecycline is currently given by slow intravenous infusion (30 to
60 minutes). A single dose of 100 mg is given first, followed by 50
mg every twelve hours after that.
[0157] Lincomycin is a lincosamide antibiotic that comes from the
actinomyces Streptomyces lincolnensis. It has been structurally
modified by thionyl chloride to its more commonly known
7-chloro-7-deoxy derivative, clindamycin. Its systematic (IUPAC)
name is
(2S,4R)--N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(met-
hylsulfanyl)oxan-2-yl]propyl]-1-methyl-4-propylpyrrolidine-2-carboxamide.
Its chemical formula is C.sub.18H.sub.34N.sub.2O.sub.6S with a
molecular mass 406.538 g/mol.
[0158] An antibiotic is a substance or compound (also called
chemotherapeutic agent) that kills or inhibits the growth of
bacteria. Antibiotics belong to the group of antimicrobial
compounds, i.e., those for treating infections caused by
microorganisms, including viruses, fungi, and protozoa. Penicillin
is a group of antibiotics derived from Penicillium fungi. They are
Beta-lactam antibiotics used in the treatment of bacterial
infections caused by susceptible, usually Gram-positive, organisms.
The term Penam is used to describe the core skeleton of a member of
a penicillin antibiotic. This skeleton has the molecular formula
R--C.sub.9H.sub.11N.sub.2O.sub.4S, where R is a variable side
chain. Ampicillin is a beta-lactam antibiotic that has been used
extensively to treat bacterial infections since 1961. It is
considered part of the aminopenicillin family and is roughly
equivalent to amoxicillin in terms of spectrum and level of
activity. It can sometimes result in non-allergic reactions that
range in severity from a rash (e.g. patients with mononucleosis) to
potentially lethal anaphylaxis. Ampicillin is closely related to
amoxicillin, another type of penicillin, and both are used to treat
urinary tract infections, otitis media, uncomplicated
community-acquired pneumonia, Haemophilus influenzae, salmonellosis
and Listeria meningitis. Other types include Penicillin V, Procaine
benzylpenicillin, and Benzathine benzylpenicillin.
[0159] Some aspects of the invention relate to a method of treating
infections caused by microorganisms in an animal subject, the
method comprising administering nanoparticles composed of an
antibiotic, chitosan, and a core portion of negatively charged
substrate, wherein a surface of the nanoparticles is dominated by
the chitosan. In one embodiment, the method of administering the
nanoparticles may comprise an oral route, intravenous route,
subcutaneous injection route, intramuscular route, buccal route,
intranasal route, parenteral route, and the like.
[0160] Piperacillin is an extended spectrum beta-lactam antibiotic
of the ureidopenicillin class. It is normally used together with a
beta-lactamase inhibitor such as tazobactam, which is commercially
available. The combination has activity against many Gram-positive
and Gram-negative pathogens and anaerobes, including Pseudomonas
aeruginosa. The systematic (IUPAC) name is
(2,5',5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]--
2-phenyl-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-
e-2-carboxylic acid. Its chemical formula is
C.sub.23H.sub.26N.sub.5O.sub.7S with a molecular mass 516.548
g/mol. Piperacillin is not absorbed orally, and must therefore be
given by intravenous or intramuscular injection;
piperacillin/tazobactam is administered intravenously every 6 or 8
hours; the drug may also be given by continuous infusion, but this
has not been shown to be superior.
[0161] An aminoglycoside is a molecule composed of a sugar group
and an amino group. Several aminoglycosides function as antibiotics
that are effective against certain types of bacteria. They include
amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin, and
apramycin.
[0162] Aminoglycosides have several potential antibiotic
mechanisms, some as protein synthesis inhibitors, although their
exact mechanism of action is not fully known: (1) They interfere
with the proofreading process, causing increased rate of error in
synthesis with premature termination; (2) Also, there is evidence
of inhibition of ribosomal translocation where the peptidyl-tRNA
moves from the A-site to the P-site; (3) They can also disrupt the
integrity of bacterial cell membrane.
[0163] Aminoglycosides bind to the bacterial 30S ribosomal subunit;
some work by binding to the 50S subunit. There is a significant
relationship between the dose administered and the resultant plasma
level in blood. Therapeutic drug monitoring (TDM) is necessary to
obtain the correct dose. These agents exhibit a post-antibiotic
effect in which there is no or very little drug level detectable in
blood, but there still seems to be inhibition of bacterial
re-growth. This is due to strong, irreversible binding to the
ribosome, and remains intracellular long after plasma levels drop.
This allows a prolonged dosage interval. Depending on their
concentration, they act as bacteriostatic or bactericidal
agents.
[0164] The protein synthesis inhibition of aminoglycosides does not
usually produce a bactericidal effect, let alone a rapid one as is
frequently observed on susceptible Gram-negative bacilli.
Aminoglycosides competitively displace cell biofilm-associated
Mg.sup.2+ and Ca.sup.2+ that link the polysaccharides of adjacent
lipopolysaccharide molecules. The result is shedding of cell
membrane blebs, with formation of transient holes in the cell wall
and disruption of the normal permeability of the cell wall. This
action alone may be sufficient to kill most susceptible
Gram-negative bacteria before the aminoglycoside has a chance to
reach the 30S ribosome.
[0165] Traditionally, the antibacterial properties of
aminoglycosides were believed to result from inhibition of
bacterial protein synthesis through irreversible binding to the 30S
bacterial ribosome. This explanation, however, does not account for
the potent bactericidal properties of these agents, since other
antibiotics that inhibit the synthesis of proteins (such as
tetracycline) are not bactericidal. Recent experimental studies
show that the initial site of action is the outer bacterial
membrane. The cationic antibiotic molecules create fissures in the
outer cell membrane, resulting in leakage of intracellular contents
and enhanced antibiotic uptake. This rapid action at the outer
membrane probably accounts for most of the bactericidal
activity.
[0166] Energy is needed for aminoglycoside uptake into the
bacterial cell. Anaerobes have less energy available for this
uptake, so aminoglycosides are less active against anaerobes.
Aminoglycosides are useful primarily in infections involving
aerobic, gram-negative bacteria, such as Pseudomonas,
Acinetobacter, and Enterobacter. In addition, some Mycobacteria,
including the bacteria that cause tuberculosis, are susceptible to
aminoglycosides. The most frequent use of aminoglycosides is
empiric therapy for serious infections such as septicemia,
complicated intraabdominal infections, complicated urinary tract
infections, and nosocomial respiratory tract infections. Usually,
once cultures of the causal organism are grown and their
susceptibilities tested, aminoglycosides are discontinued in favor
of less toxic antibiotics.
[0167] Streptomycin was the first effective drug in the treatment
of tuberculosis, though the role of aminoglycosides such as
streptomycin and amikacin has been eclipsed (because of their
toxicity and inconvenient route of administration) except for
multiple drug resistant strains.
[0168] Infections caused by gram-positive bacteria can also be
treated with aminoglycosides, but other types of antibiotics are
more potent and less damaging to the host. In the past, the
aminoglycosides have been used in conjunction with beta-lactam
antibiotics in streptococcal infections for their synergistic
effects, particularly in endocarditis. One of the most frequent
combinations is ampicillin (a beta-lactam, or penicillin-related
antibiotic) and gentamicin. Often, hospital staff refer to this
combination as "amp and gent" or more recently called "pen and
gent" for penicillin and gentamicin. Aminoglycosides are mostly
ineffective against anaerobic bacteria, fungi and viruses.
[0169] Experimentation with aminoglycosides as a treatment of
cystic fibrosis (CF) has shown some promising results. CF is caused
by a mutation in the gene coding for the cystic fibrosis
transmembrane conductance regulator (CFTR) protein. In
approximately 10% of CF cases the mutation in this gene causes its
early termination during translation, leading to the formation of
is truncated and non-functional CFTR protein. It is believed that
gentamicin distorts the structure of the ribosome-RNA complex,
leading to a misreading of the termination cordon, causing the
ribosome to "skip" over the stop sequence and to continue with the
normal elongation and production of the CFTR protein. The treatment
is still experimental but showed improvement in cells from CF
patients with susceptible mutations.
[0170] Since they are not absorbed from the gut via conventional
transcellular permeability, they are administered intravenously and
intramuscularly. Some are used in topical preparations for wounds.
Oral administration can be used for gut decontamination (e.g. in
hepatic encephalopathy). Tobramycin may be administered in a
nebulized form. Some aspects of the invention relate to a
nanoparticle formulation as disclosed herein to enhance absorption
via paracellular permeation route.
[0171] Temsirolimus is an intravenous drug for the treatment of
renal cell carcinoma (RCC), developed by Wyeth Pharmaceuticals and
approved by the FDA in late May 2007. It is a derivative of
sirolimus and is sold as Torisel. Its chemical formula is
C.sub.56H.sub.87NO.sub.16 with a molecular mass 1030.28 g/mol. mTOR
(mammalian target of rapamycin) is a kinase enzyme inside the cell
that collects and interprets the numerous and varied growth and
survival signals received by tumor cells. When the kinase activity
of mTOR is activated, its downstream effectors, the synthesis of
cell cycle proteins such as cyclin D and hypoxia-inducible
factor-1a (HIF-1a) are increased. HIF-1a then stimulates VEGF.
Temsirolimus is a specific inhibitor of mTOR and interferes with
the synthesis of proteins that regulate proliferation, growth, and
survival of tumor cells. Treatment with temsirolimus leads to cell
cycle arrest in the G1 phase, and also inhibits tumor angiogenesis
by reducing synthesis of VEGF. The recommended dose of temsirolimus
is 25 mg IV infused over 30-60 minutes once per week.
[0172] Some aspects of the invention relate to a method of
enhancing epithelial permeation of bioactive agents configured and
adapted for delivering at least one bioactive agent in an animal
subject comprising administering nanoparticles composed of
.gamma.-PGA and chitosan, wherein the nanoparticles are loaded with
a therapeutically effective amount or dose of the at least one
bioactive agent. The nanoparticle of the present invention is an
effective intestinal delivery system for peptide and protein drugs
and other large hydrophilic molecules. In a further embodiment, the
bioactive agent is selected from the group consisting of proteins,
peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, antiepileptic drug, and
anti-inflammatory drugs. In a further embodiment, the bioactive
agent is selected from the group consisting of calcitonin,
cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin
releasing hormone (TRH), follicle stimulating hormone (FSH),
luteinizing hormone (LH), vasopressin and vasopressin analogs,
catalase, superoxide dismutase, interleukin-Il (IL2),
interleukin-11 (IL-11), interferon, colony stimulating factor
(CSF), tumor necrosis factor (TNF) and melanocyte-stimulating
hormone.
[0173] In a further embodiment, the bioactive agent is an Alzheimer
antagonist. In one embodiment, the antiepileptic drug may include
Neurontin (gabapentin), Lamictal (lamotrigine), Febatol
(felbamate), Topamax (topiramate), Cerebyx (fosphenyloin), Dilantin
(phenyloin), Depakene (valproic acid), Tegretol (carbamazepine),
carbamazepine epoxide, Vimpat (lacosamide) and phenobarbitol.
Fosphenyloin (Cerebyx by Parke-Davis; Prodilantin by Pfizer Holding
France) is a water-soluble phenyloin prodrug used only in hospitals
for the treatment of epileptic seizures through parental delivery.
Fosphenyloin has systematic (IUPAC) name of
(2,5-dioxo-4,4-diphenyl-imidazolidin-1-yl)methoxyphosphonic acid.
It has the chemical formula of C.sub.16H.sub.15NO.sub.6P with
molecular mass 362.274 g/mol.
Example No. 17
Nanoparticles Loaded with Heparin
[0174] Heparin is a negatively charged drug that serves
therapeutically as an anti-coagulant. Heparin is generally
administered by intravenous injection. Some aspects of the
invention relate to heparin nanoparticles for oral administration
or subcutaneous administration. In a further embodiment, heparin
serves as at least a portion of the core substrate with chitosan as
shell substrate, wherein heparin conjugates with at least one
bioactive agent as disclosed herein. In preparation, nanoparticles
were obtained upon addition of heparin Leo aqueous solution (2
nil), using a pipette (0.5-5 ml, PLASTIBRAND.RTM., BrandTech
Scientific Inc., Germany), into a low-MW CS aqueous solution (pH
6.0, 10 ml) with excess concentrations under magnetic stirring at
room temperature. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. Heparin is wholly or
substantially totally encapsulated within the nanoparticles. The
nanoparticles thus obtained via the simple and mild ionic-gelation
method described herein show typical characteristics in a
spheroidal configuration with a particle size of between about 50
to 400 nm, a positive surface charge and a narrow polydispersity
index. Table 4 shows the conditions of solution preparation and the
average nanoparticle size.
TABLE-US-00005 TABLE 4 Heparin conc. Chitosan conc. Particle
Conditions @2 ml @10 ml size (nm) A 200 iu/ml 0.09% 298.2 .+-. 9.3
B 100 iu/ml 0.09% 229.1 .+-. 4.5 C 50 iu/ml 0.09% 168.6 .+-. 1.7 D
25 iu/ml 0.09% 140.1 .+-. 2.3
[0175] To evaluate the pH stability of the heparin-containing
nanoparticles from Example no. 17, the nanoparticles from Condition
D in Table 4 are subjected to various pH level for 2 hours (sample
size=7). Table 5 shows the average size, size distribution
(polydispersity index: PI) and zeta potential (Zeta) of the
nanoparticles at the end of 2 hours under various pH environments.
The data shows the nanoparticles are relatively stable. In one
embodiment, the nanoparticles of the present invention may include
heparin, heparin sulfate, small molecular weight heparin, and
heparin derivatives.
TABLE-US-00006 TABLE 5 pH 1.5 2.6 6.6 7.4 Deionized water @5.9 Size
(nm) 150 .+-. 9 160 .+-. 12 153 .+-. 2 154 .+-. 4 147 .+-. 5 PI
0.54 .+-. 0.03 0.50 .+-. 0.04 0.08 .+-. 0.02 0.32 .+-. 0.03 0.37
.+-. 0.02 Zeta (+) 15 .+-. 2 33 .+-. 6 15 .+-. 0.1 11 .+-. 0.2 18
.+-. 4
[0176] In a further embodiment, a pharmaceutically effective amount
of growth factor such as bFGF is added to heparin Leo aqueous
solution before the pipetting step in Example No. 15. In our
laboratory, growth factors and proteins with pharmaceutically
effective amounts have been successfully conjugated with heparin to
form nanoparticles of the present invention with chitosan as the
shell substrate, wherein the growth factor is selected from the
group consisting of Vascular Endothelial Growth Factor (VEGF),
Vascular Endothelial Growth Factor 2 (VEGF2), basic Fibroblast
Growth Factor (bFGF), Vascular Endothelial Growth Factor 121
(VEGF121), Vascular Endothelial Growth Factor 165 (VEGF165),
Vascular Endothelial Growth Factor 189 (VEGF189), Vascular
Endothelial Growth Factor 206 (VEGF206), Platelet Derived Growth
Factor (PDGF), Platelet Derived Angiogenesis Factor (PDAF),
Transforming Growth Factor-.beta. (TGF-.beta.), Transforming Growth
Factor-.alpha. (TGF-.alpha.), Platelet Derived Epidermal Growth
Factor (PDEGF), Platelet Derived Wound Healing Formula (PDWHF),
epidermal growth factor, insulin-like growth factor, acidic
Fibroblast Growth Factor (aFGF), human growth factor, and
combinations thereof; and the protein is selected from the group
consisting of haemagglutinin (HBHA), Pleiotrophin, buffalo seminal
plasma proteins, and combinations thereof.
[0177] In a co-pending application, U.S. patent application Ser.
No. 10/916,170 filed Aug. 11, 2004, it is disclosed that a
biomaterial with free amino groups of lysine, hydroxylysine, or
arginine residues within biologic tissues is crosslinkable with
genipin, a crosslinker (Biomaterials 1999; 20:1759-72). It is also
disclosed that the crosslinkable biomaterial may be crosslinked
with a crosslinking agent or with light, such as ultraviolet
irradiation, wherein the crosslinkable biomaterial may be selected
from the group consisting of collagen, gelatin, elastin, chitosan,
NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biological
sealant, and the like. Further, it is disclosed that a crosslinking
agent may be selected from the group consisting of genipin, its
derivatives, analog (for example, aglycon geniposidic acid),
stereoisomers and mixtures thereof. In one embodiment, the
crosslinking agent may further be selected from the group
consisting of epoxy compounds, dialdehyde starch, glutaraldehyde,
formaldehyde, dimethyl suberimidate, carbodiimides, succinimidyls,
diisocyanates, acyl azide, reuterin, ultraviolet irradiation,
dehydrothermal treatment, tris(hydroxymethyl)phosphine,
ascorbate-copper, glucose-lysine and photo-oxidizers, and the
like.
[0178] In one embodiment, it is disclosed that loading a drug onto
a chitosan-containing biological material crosslinked with genipin
or other crosslinking agent may be used as biocompatible drug
carriers for drug slow-release or sustained release. Several
biocompatible plastic polymers or synthetic polymers have one or
more amine group in their chemical structures, for example
poly(amides) or poly(ester amides). The amine group may become
reactive toward a crosslinking agent, such as glutaraldehyde,
genipin or epoxy compounds of the present invention. In one
embodiment, the nanoparticles comprised of crosslinkable
biomaterial is crosslinked, for example up to about 50% degree or
more of crosslinking, preferably about 1 to about 20% degree of
crosslinking of the crosslinkable components of the biomaterial,
enabling sustained biodegradation of the biomaterial and/or
sustained drug release.
[0179] By modifying the chitosan structure to alter its charge
characteristics, such as grafting the chitosan with EDTA, methyl,
N-trimethyl, alkyl (for example, ethyl, propyl, butyl, isobutyl,
etc.), polyethylene glycol (PEG), or heparin (including low
molecular weight heparin, regular molecular weight heparin, and
genetically modified heparin), the surface charge density (zeta
potential) of the CS-.gamma. PGA nanoparticles may become more pH
resistant or hydrophilic. In one embodiment, the chitosan is
grafted with polyacrylic acid. In one embodiment, the chitosan
employed is N-trimethyl chitosan (TMC), low MW-chitosan,
EDTA-chitosan, chitosan derivatives, and/or combinations thereof.
An exemplary chemical structure for EDTA-chitosan is shown
below:
##STR00003##
[0180] By way of illustration, trimethyl chitosan chloride might be
used in formulating the CS-.gamma. PGA nanoparticles for
maintaining its spherical biostability at a pH lower than 2.5,
preferably at a pH as low as 1.0. Some aspects of the invention
provide a drug-loaded chitosan-containing biological material
crosslinked with genipin or other crosslinking agent as a
biocompatible drug carrier for enhancing biostability at a pH lower
than 2.5, preferably within at a pH as low as 1.0.
[0181] It is known that the pKa values of CS (amine groups) and
.gamma.-PGA (carboxylic groups) are 6.5 and 2.9, respectively. NPs
were prepared in DI water (pH 6.0). At pH 6.0, CS (TMC25) and
.gamma.-PGA were ionized. The ionized CS (TMC25) and .gamma.-PGA
could form polyelectrolyte complexes, which resulted in a matrix
structure with a spherical shape. At pH 1.2-2.0, most carboxylic
groups on .gamma.-PGA were in the form of --COOH. Hence, there was
little electrostatic interaction between CS (TMC25) and
.gamma.-PGA; thus NPs became disintegrated (Table 6). Similarly, at
pH values above 6.6, the free amine groups on CS (TMC25) were
deprotonated; thus leading to the disintegration of NPs. This might
limit the efficacy of drug delivery and absorption in the small
intestine.
[0182] When increasing the degree of quaternization on TMC (TMC40
and TMC55), the stability of NPs in the pH range of 6.6-7.4
increased significantly. However, the swelling of TMC55/.gamma.-PGA
NPs at pH 7.4 was minimal (due to the highly quaternized TMC55),
which might limit the release of loaded drugs. In contrast,
TMC40/.gamma.-PGA NPs swelled significantly with increasing the pH
value. TMC40/.gamma.-PGA NPs (collapsed NPs or fragments) still
retained a positive surface charge with a zeta potential value of
17.3 mV at pH 7.4.
[0183] Thus, TMC40/.gamma.-PGA/drug NPs have superior stability in
a broader pH range compared to CS/.gamma.-PGA/drug NPs. In one
embodiment, at around body fluid pH of about 7.4, the bioactive
nanoparticles of the present invention may appear to be in
configuration of chitosan-shelled fragments or chitosan-containing
fragments. At least a portion of the surface of the
chitosan-shelled fragments or chitosan-containing fragments from
the bioactive nanoparticles of the present invention shows positive
zeta potential characteristics.
[0184] The results of molecular dynamic simulations showed that the
molecular chains of TMC40 (in dark black) and .gamma.-PGA (in light
black) in their self-assembled complex were tightly entangled with
each other at pH 6.0. The surface of the complex was dominated by
TMC40 molecules. Relaxations of TMC40 and .gamma.-PGA molecular
chains at pH 2.5 resulted in a moderate swelling of the
TMC40/.gamma.-PGA complex, while its surface was still dominated by
the positively charged TMC molecules, thus retaining a positive
surface charge.
[0185] Similarly, relaxations of TMC40 and .gamma.-PGA molecular
chains at pH 7.4 resulted in a significant swelling of the
TMC40/.gamma.-PGA complex, while its surface was still dominated by
the positively charged TMC molecules, thus retaining a positive
surface charge. The swollen TMC40/.gamma.-PGA/drug nanoparticles
tend to slightly disintegrate (due to the effect of its pH
instability) so to form fragments consisting of
TMC40/.gamma.-PGA/drug with surface-dominated TMC40.
[0186] The TMC40/.gamma.-PGA/drug fragments with surface-dominated
TMC40 would adhere and infiltrate into the mucus of the epithelial
membrane of the blood-brain barrier, and then trigger transiently
opening the tight junctions between enterocytes. Table 6 shows mean
particle sizes, zeta potential values, and polydispersity indices
of nanoparticles (NPs) self-assembled by TMC polymers with
different degrees of quaternization and .gamma.-PGA at distinct pH
environments (n=5 batches). As shown in Table 6, TMC40/.gamma.-PGA
NPs still retained a positive surface charge with a zeta potential
value of 17.3 mV at 7.4.
[0187] Freeze-Dried Nanoparticles
[0188] A pharmaceutical composition of nanoparticles of the present
invention may comprise a first component of at least one bioactive
agent, a second component of chitosan (including regular molecular
weight and low molecular weight chitosan), and a third component
that is negatively charged. In one embodiment, the second component
dominates on a surface of the nanoparticle. In another embodiment,
the chitosan is N-trimethyl chitosan.
[0189] In still another embodiment, the low molecular weight
chitosan has a molecular weight less than that of a regular
molecular weight chitosan. The nanoparticles may further comprise
tripolyphosphate and magnesium sulfate. For example, a first
solution of (2 ml 0.1% .gamma.-PGA aqueous solution @pH 7.4+0.05%
Insulin+0.1% Tripolyphosphate (TPP)+0.2% MgSO4) is added to a base
solution (10 ml 0.12% chitosan aqueous solution @pH 6.0) as
illustrated in Example no. 3 under magnetic stirring at room
temperature. Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour. The bioactive agent, the third component,
tripolyphosphate and magnesium sulfate are wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water for
freeze-drying preparation. Other operating conditions or other
bioactive agent (such as protein, peptide, siRNA, growth factor,
the one defined and disclosed herein, and the like) may also
apply.
[0190] Several conventional coating compounds that form a
protective layer on particles are used to physically coat or mix
with the nanoparticles before a freeze-drying process. The coating
compounds may include trehalose, mannitol, glycerol, and the like.
Trehalose, also known as mycose, is an alpha-linked (disaccharide)
sugar found extensively but not abundantly in nature. It can be
synthesized by fungi, plants and invertebrate animals. It is
associated with anhydrobiosis--the ability of plants and animals to
withstand prolonged periods of desiccation. The sugar is thought to
form a gel phase as cells dehydrate, which prevents disruption of
internal cell organelles by effectively splinting them in position.
Rehydration then allows normal cellular activity to resume without
the major, generally lethal damage, which would normally follow a
dehydration/rehydration cycle. Trehalose has the added advantage of
being an antioxidant.
[0191] Trehaloze has a chemical formula as
C.sub.12H.sub.22O.sub.11.2H.sub.2O. It is listed as CAS no. 99-20-7
and PubChem 7427. The molecular structure for trehalose is shown
below.
##STR00004##
[0192] Trehalose was first isolated from the ergot of rye.
Trehalose is a non-reducing sugar formed from two glucose units
joined by a 1-1 alpha bond, giving it the name
.alpha.-D-glucopyranosyl-(1.fwdarw.1)-.alpha.-D-glucopyranoside.
The bonding makes trehalose very resistant to acid hydrolysis, and
therefore stable in a solution at high temperatures, even under
acidic conditions. The bonding also keeps non-reducing sugars in a
closed-ring form, such that the aldehyde or ketone end-groups do
not bind to the lysine or arginine residues of proteins (a process
called glycation). Trehalose has about 45% the sweetness of
sucrose. Trehalose is less soluble than sucrose, except at high
temperatures (>80.degree. C.). Trehalose forms a rhomboid
crystal as the dihydrate, and has 90% of the calorific content of
sucrose in that form. Anhydrous forms of trehalose readily regain
moisture to form the dihydrate. Trehalose has also been used in at
least one biopharmaceutical formulation, the monoclonal antibody
trastuzumab, marketed as Herceptin. It has a solubility of 68.9
g/100 g H.sub.2O at 20.degree. C.
[0193] Mannitol or hexan-1,2,3,4,5,6-hexyl
(C.sub.6H.sub.8(OH).sub.6) is an osmotic diuretic agent and a weak
renal vasodilator. Chemically, mannitol is a sugar alcohol, or a
polyol; it is similar to xylitol or sorbitol. However, mannitol has
a tendency to lose a hydrogen ion in aqueous solutions, which
causes the solution to become acidic. For this reason, it is not
uncommon to add a substance to adjust its pH, such as sodium
bicarbonate. Mannitol has a chemical formula
C.sub.5H.sub.14O.sub.6. It is listed as CAS no. 69-65-8 and PubChem
453. The molecular structure for mannitol is shown below.
##STR00005##
[0194] Glycerol is a chemical compound with the formula
HOCH.sub.2CH(OH)CH.sub.2OH. This colorless, odorless, viscous
liquid is widely used in pharmaceutical formulations. Commonly
called glycerin or glycerine, it is a sugar alcohol and is
fittingly sweet-tasting with low toxicity. Glycerol has three
hydrophilic alcoholic hydroxyl groups that are responsible for its
solubility in water and its hygroscopic nature. Glycerol has a
chemical formula as C.sub.3H.sub.5(OH).sub.3. It is listed as CAS
no. 56-81-5. The molecular structure for glycerol is shown
below.
##STR00006##
Example No. 18
Freeze-Drying Process for Nanoparticles
[0195] Each nanoparticles (at 2.5% concentration) were mixed with a
solution of four types of liquid at a 1:1 volume ratio for about 30
minutes until fully dispersed. The mixed particle-liquid was then
freeze-dried under a lyophilization condition, for example, at
about -80.degree. C. and <25 mmHg pressure for about 6 hours.
The parameters in a selected lyophilization condition may vary
slightly from the aforementioned numbers. The four types of liquid
used in the experiment include: (A) DI water; (B) trehalose; (C)
mannitol; and (D) glycerol, whereas the concentration of the liquid
(A) to liquid (C) in the solution was set at 2.5%, 5% and 10%.
After a freeze-drying process, the mixed particle-liquid was
rehydrated with DI water at a 1:5 volume ratio to assess the
integrity of nanoparticles in each type of liquid. The results are
shown in Table 7. By comparing the particle size, polydispersity
index, and zeta-potential data, only the nanoparticles from the
freeze-dried particle-trehalose runs (at 2.5%, 5%, and 10%
concentration level) show comparable properties to those of the
pre-lyophilization nanoparticles. Under the same data analysis, the
nanoparticles from the freeze-dried particle-mannitol runs (at
2.5%, and 5% concentration level) show somewhat comparable
properties to those of the pre-lyophilization nanoparticles.
TABLE-US-00007 TABLE 6 Parameters of nanoparticles (NPs)
self-assembled by TMC polymers with different degrees of
quaternization. Mean Particle Zeta Potential Polydispersity Size
(nm) (mV) Index CS/.gamma.-PGA NPs pH 1.2 N/A N/A 1 pH 2.0 N/A N/A
1 pH 2.5 113.3 .+-. 1.6 38.6 .+-. 0.8 0.14 .+-. 0.01 pH 6.0 104.1
.+-. 1.2 36.2 .+-. 2.5 0.11 .+-. 0.02 pH 6.6 245.6 .+-. 4.5 12.9
.+-. 0.4 0.17 .+-. 0.11 pH 7.0 N/A N/A 1 pH 7.4 N/A N/A 1
TMC25/.gamma.-PGA NPs pH 1.2 N/A N/A 1 pH 2.0 N/A N/A 1 pH 2.5
396.4 .+-. 4.7 32.1 .+-. 1.6 0.32 .+-. 0.11 pH 6.0 101.3 .+-. 3.1
30.9 .+-. 2.1 0.13 .+-. 0.04 pH 6.6 N/A N/A 1 pH 7.0 N/A N/A 1 pH
7.4 N/A N/A 1 TMC40/.gamma.-PGA NPs pH 1.2 N/A N/A 1 pH 2.0 N/A N/A
1 pH 2.3 272.2 .+-. 2.3 38.6 .+-. 2.7 0.25 .+-. 0.23 pH 2.5 252.4
.+-. 3.5 35.4 .+-. 1.1 0.21 .+-. 0.04 pH 6.0 106.3 .+-. 2.3 32.3
.+-. 2.1 0.15 .+-. 0.14 pH 6.6 238.3 .+-. 3.1 24.3 .+-. 1.4 0.09
.+-. 0.03 pH 7.0 296.7 .+-. 4.7 20.4 .+-. 0.3 0.18 .+-. 0.11 pH 7.4
498.4 .+-. 6.8 17.3 .+-. 0.6 0.38 .+-. 0.21 TMC55/.gamma.-PGA NPs
pH 1.2 N/A N/A 1 pH 2.0 252.5 .+-. 4.1 35.6 .+-. 4.2 0.16 .+-. 0.08
pH 2.5 221.4 .+-. 3.5 32.5 .+-. 3.4 0.15 .+-. 0.02 pH 6.0 114.6
.+-. 2.3 30.6 .+-. 3.8 0.12 .+-. 0.03 pH 6.6 141.2 .+-. 1.6 24.8
.+-. 3.4 0.15 .+-. 0.02 pH 7.0 144.6 .+-. 4.8 20.4 .+-. 1.7 0.18
.+-. 0.14 pH 7.4 141.2 .+-. 0.9 18.9 .+-. 4.1 0.11 .+-. 0.11 N/A:
Precipitation of aggregates was observed.
TABLE-US-00008 TABLE 7 Properties of nanoparticles before and after
an exemplary freeze-drying process. (A: before a freeze-drying
process NPs solution Conc. 2.50% Size(mm) 266 Kcps 352.2 PI 0.291
Zeta Potential 25.3 (B: after a freeze-drying process) A: DI Water
B: Trehalose C: Mannitol D: Glycerol DI water + NPs Trehalose + NPs
Mannitol + NPs Glycerol + NPs (volume 1:1) (volume 1:1) (volume
1:1) (volume 1:1) Conc. 2.50% 5.00% 10.00% 2.50% 5.00% 2.50% 5.00%
10.00% Size(mm) 9229.1 302.4 316.7 318.9 420.1 487.5 6449.1 7790.3
1310.5 Kcps 465.3 363.7 327.7 352.2 305.4 303.7 796.1 356.1 493.3
PI 1 0361 0.311 0.266 0.467 0.651 1 1 1 Zeta Potential 25.6 24.6
24.7 24.4 25.3
[0196] FIG. 16 shows an illustrative mechanism of nanoparticles
released from the enteric-coated capsules. FIG. 16(A) shows the
phase where nanoparticles are in the gastric cavity, wherein the
freeze-dried nanoparticles 82 are encapsulated within an initial
enteric coating or coated capsule 81. FIG. 16(B) shows a schematic
of the nanoparticles during the phase of entering small intestine,
wherein the enteric coat and its associated capsule starts to
dissolve 83 and a portion of nanoparticles 82 is released from the
capsule and contacts fluid. FIG. 16(C) shows the phase of
nanoparticles in the intestinal tract, wherein the nanoparticles
revert to a wet state having chitosan at its surface. In an
alternate embodiment, nanoparticles may be released from
alginate-calcium coating. In preparation, nanoparticles are first
suspended in a solution that contains calcium chloride, wherein the
calcium ions are positively charged. With a pipette, alginate with
negatively charged carboxyl groups is slowly added to the calcium
chloride solution. Under gentle stirring, the alginate-calcium
starts to conjugate, gel, and coat on the nanoparticle surface. In
simulated oral administration of the alginate-calcium coated
nanoparticles, nanoparticles start to separate from the coating
when they enter the small intestines.
Example No. 19
Freeze-Dried Nanoparticles in Animal Evaluation
[0197] In the in vivo study, rats as prepared and conditioned
according to Example no. 10 were used in this evaluation. In the
animal evaluation study, diabetic rats fasted for 12 hours and were
then subjected to three different conditions: (a) oral deionized
water (DI) administration as negative control; (b) oral
insulin-loaded lyophilized nanoparticles administration, whereas
the nanoparticles have an insulin loading content of 4.4% with an
insulin loading efficiency of 48.6% and are loaded in a capsule
with a surface enteric coating; and (c) subcutaneous (SC) insulin
injection at 5 U/kg as positive control. The blood glucose
concentration from rat's tail was measured over time.
[0198] FIG. 17 shows glucose change (hypoglycemic index) versus
time of the in vivo animal study (n=5). The glucose change as a
percentage of base lines for oral DI administration (control) over
a time interval of 10 hours appears relatively constant within the
experimental measurement error range. As anticipated, the glucose
decrease for the SC insulin injection method is evident in rat
blood at a very early time interval starts to taper off after 2
hours, and ends at about 6 hours in this exemplary study. The most
important observation of the study comes from the oral
administration route with insulin-loaded lyophilized (namely,
freeze-dried) nanoparticles. Nanoparticles of this example have an
insulin LC at 4.4%, whereas nanoparticles from Example no. 10 had
an insulin LC at 14.1% in FIG. 14). With the same amount of
nanoparticles in both examples, the insulin-feeding ratio of
Example no. 19 to Example no. 10 is about 1:3. In other words, the
insulin fed to a rat in this study from nanoparticles is about 1/3
of the insulin from nanoparticles fed to rats in Example no.
10.
[0199] The blood glucose begins to decrease from the base line at
about 3 hours after administration and sustains a lower glucose
level for more than 10 hours into study. It implies that the
current insulin-loaded nanoparticles may modulate the glucose level
in animals in a sustained or prolonged effective mode. Some aspects
of the invention provide a method of treating diabetes of an animal
subject comprising orally administering insulin-containing
nanoparticles with a dosage effective amount of the insulin to
treat the diabetes, wherein at least a portion of the nanoparticles
comprises a positively charged shell substrate and a negatively
charged core substrate. In one embodiment, the dosage effective
amount of the insulin to treat diabetes comprises an insulin amount
of between about 15 units to 45 units per kilogram body weight of
the animal subject, preferably 20 to 40 units, and most preferably
at about 25 to 35 units insulin per kilogram body weight. In one
embodiment, the lyophilized nanoparticles may be fed as-is to an
animal without being loaded in an enterically coated capsule.
Example No. 20
Nanoparticles Loaded with Enhanced Insulin Loading
[0200] In a co-pending application, U.S. patent application Ser.
No. 11/881,185 filed Jul. 26, 2007, entire contents of which are
incorporated herein by reference, it is disclosed that a novel
nanoparticle may comprise a shell substrate of chitosan and a core
substrate consisting of at least one bioactive agent, MgSO.sub.4,
TPP, and a negatively charged substrate that is neutralized with
chitosan in the core. FIG. 18 shows insulin-loaded nanoparticles
with a core composition comprised of .gamma.-PGA, MgSO.sub.4,
sodium tripolyphosphate (TPP), and insulin. Nanoparticles were
obtained upon addition of core component, using a pipette (0.5-5
ml, PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
CS aqueous solution (pH 6.0, 10 ml) at certain concentrations under
magnetic stirring at room temperature. Nanoparticles were collected
by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were
discarded and nanoparticles were resuspended in deionized water for
further studies. In one embodiment, nanoparticles are encapsulated
in a gelcap, or are lyophilized before being loaded in a gelcap or
in a tablet. The nanoparticles thus obtained via the simple and
mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge and a
narrow polydispersity index. The sodium tripolyphosphate has a
chemical formula of Na.sub.5P.sub.3O.sub.10 as shown below:
##STR00007##
[0201] In the example, the core composition may be varied and
evaluated with a preferred composition of 2 ml .gamma.-PGA aqueous
solution at pH 7.4 with insulin, MgSO.sub.4 and TPP, resulting in a
ratio of CS:.gamma.-PGA:TPP:MgSO4:insulin=6.0:1.0:1.0:2.0:0.05.
Thus, the nanoparticles show characteristics as disclosed herein
with a chitosan shell and a core composition consisting of
.gamma.-PGA, MgSO.sub.4, TPP, and insulin and have an average
loading efficiency of 72.8% insulin and an average loading content
of 21.6% insulin.
[0202] In the enhanced drug loading of the present example, there
provides two or more distinct ionic crosslink mechanisms. In one
embodiment, the nanoparticles of the present invention may have a
structure or matrix of interpenetrated ionic-crosslinks (that is,
elongate ionic-crosslink chains) including a first ionic-crosslink
chain of NH.sub.3.sup.+ of CS with COO.sup.- of .gamma.-PGA, a
second ionic-crosslink chain of NH.sub.3.sup.+ of CS with
SO.sub.4.sup.2- of MgSO.sub.1, a third ionic-crosslink chain of
Mg.sup.2+ of MgSO.sub.4 with COO.sup.- of .gamma.-PGA, and/or a
fourth ionic-crosslink chain of Na.sub.3P.sub.3O.sub.10.sup.2- of
TPP with NH.sub.3.sup.+ of CS or Mg.sup.2+ of MgSO.sub.4.
[0203] Some aspects of the invention relate to a nanoparticle
composition for oral administration with an insulin loading
efficiency and content at higher than 45% and 14% (preferably up to
about 73% and 22%), respectively. The prepared nanoparticles (NPs)
are stable at a pH range of 2.0 to 7.1. This broad range allows the
chitosan-shelled nanoparticle to be temporarily stable in most of
the intestine region (including duodenum, jejunum, and ileum) for
enhanced membrane absorption and permeability of active ingredient
(for example, insulin, exenatide or pramlintide). Some aspects of
the invention provide a chitosan-shelled nanoparticle with a core
composition of .gamma.-PGA, MgSO.sub.4, TPP, and at least one
bioactive agent, such as insulin, exenatide, or pramlintide for
treatment of diabetes. In an alternate embodiment, some aspects of
the invention provide a chitosan-shelled nanoparticle with a core
composition consisted of .gamma.-PGA, MgSO.sub.4, TPP, and at least
one bioactive agent. In one embodiment, negatively charged
.gamma.-PGA may conveniently be substituted by another negatively
charge substrate, such as heparin. In an experiment following the
experimental conditions of Example no. 20 by substituting insulin
with exenatide, chitosan-shelled nanoparticles with a core
composition of .gamma.-PGA, MgSO.sub.4, TPP, and exenatide exhibit
similar physical and mechanical properties compared to the ones
with insulin.
[0204] FIG. 19 shows an in vivo subcutaneous study using insulin
injectables and insulin-containing nanoparticles. The
insulin-containing nanoparticles exhibit different pharmacodynamics
and/or pharmacokinetics in a sustained releasing manner. Some
aspects of the invention relate to a pharmaceutical composition of
nanoparticles for subcutaneous or blood vessel administration in an
animal subject, the nanoparticles comprising a shell portion that
is dominated by positively charged chitosan, a core portion that
contains negatively charged substrate, wherein the negatively
charged substrate is at least partially neutralized with a portion
of the positively charged chitosan in the core portion, and at
least one bioactive agent loaded within the nanoparticles.
[0205] Some aspects of the invention relate to a method of
delivering a bioactive agent to blood circulation in an animal
subject, comprising: (a) providing nanoparticles according to a
preferred embodiment of the pharmaceutical composition of the
present invention, wherein the nanoparticles are formed via a
simple and mild ionic-gelation method; (b) administering the
nanoparticles orally toward the intestine of the animal subject via
stomach; (c) urging the nanoparticles to be absorbed onto a surface
of an epithelial membrane of the intestine via muco-adhesive
chitosan-shelled nanoparticles; (d) permeating bioactive agent to
pass through an epithelial barrier of the intestine; and (e)
releasing the bioactive agent into the blood circulation. In one
embodiment, the bioactive agent is selected from the group
consisting of exenatide, pramlintide, insulin, insulin analog, and
combinations thereof. In another embodiment, the bioactive agent
permeates through the tight junctions of the epithelial membrane
when chitosan-shelled nanoparticles break up and release the
bioactive agent at vicinity of the tight junctions.
[0206] Some aspects of the invention relate to a method for
inducing a redistribution of tight junctions' ZO-1 protein, leading
to a translocation of the ZO-1 protein to the cytoskeleton that
accompanies increased permeation in an animal subject, the method
comprising administering bioactive nanoparticles into the animal
subject with an effective dosage to induce the redistribution,
wherein the bioactive nanoparticles comprise a shell substrate of
chitosan and a core substrate that comprises poly(glutamic acid)
and the bioactive agent that is selected from the group consisting
of exenatide, pramlintide, insulin, insulin analog, and
combinations thereof.
[0207] Nanoparticles Loaded with Tumor Necrosis Factor
Inhibitors
[0208] Tumor necrosis factor (TNF) promotes an inflammatory
response, which in turn causes many of the clinical problems
associated with autoimmune disorders such as rheumatoid arthritis,
ankylosing spondylitis, Crohn's disease, psoriasis, and refractory
asthma. These disorders are sometimes treated by using a TNF
inhibitor. This inhibition can be achieved with a monoclonal
antibody such as infliximab (Remicade) or adalimumab (Humira), or
with a circulating receptor fusion protein such as etanercept
(Enbrel). Another example is pentoxifylline.
[0209] This potential applicability of anti-TNF therapies in the
treatment of rheumatoid arthritis (RA) is based on the recognition
of the role of TNF-alpha is the "master regulator" of the
inflammatory response in many organ systems. TNF and the effects of
TNF are also inhibited by a number of natural compounds, including
curcumin (a compound present in turmeric) and catechins (in green
tea). Tumor necrosis factor-alpha (TNF.alpha.) is a cytokine
produced by monocytes and macrophages, two types of white blood
cells. It mediates the immune response by increasing the transport
of white blood cells to sites of inflammation, and through
additional molecular mechanisms that initiate and amplify
inflammation.
[0210] Adalimumab (brand name HUMIRA) is a TNF inhibitor, after
infliximab and etanercept, to be approved in the United States.
Like infliximab and etanercept, adalimumab binds to TNF.alpha.,
preventing it from activating TNF receptors. Adalimumab is
constructed from a fully human monoclonal antibody, while
infliximab is a mouse-human chimeric antibody, and etanercept is a
TNF receptor-IgG fusion protein. TNF.alpha. inactivation has proven
to be important in downregulating the inflammatory reactions
associated with autoimmune diseases. As of 2008, adalimumab has
been approved by the FDA for the treatment of rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis, Crohn's disease,
moderate to severe chronic psoriasis, and juvenile idiopathic
arthritis.
[0211] Adalimumab has a chemical formula of
C.sub.6428H.sub.9912N.sub.1694O.sub.1987S.sub.46 and a molecular
mass of 144190.3 g/mol. Humira (brand name is an abbreviation of
"Human Monoclonal Antibody in Rheumatoid Arthritis") is marketed as
a subcutaneously injected treatment, typically by the patient at
home. It cannot be administered orally, because the digestive
system would destroy the drug in its current state, unless the drug
is encapsulated in nanoparticles of the present invention.
[0212] Etanercept (Enbrel) is a recombinant-DNA drug made by
combining two proteins (a fusion protein). It links human soluble
TNF receptor to the Fc component of human immunoglobulin G1 (IgG1)
and acts as a TNF inhibitor. Etanercept has a chemical formula of
C.sub.2224H.sub.3475N.sub.621O.sub.698S.sub.36 and a molecular mass
of 51234.9 g/mol. Etanercept binds to TNF.alpha. and decreases its
role in disorders involving excess inflammation in humans and other
animals, including autoimmune diseases such as ankylosing
spondylitis, juvenile rheumatoid arthritis, psoriasis, psoriatic
arthritis, rheumatoid arthritis, and, potentially a variety of
other disorders mediated by excess TNF.alpha..
[0213] Infliximab (brand name Remicade) is a drug used to treat
autoimmune disorders. Infliximab is known as a "chimeric monoclonal
antibody" (the term "chimeric" refers to the use of both mouse
(murine) and human components of the drug (i.e. murine binding
F.sub.ab domains and human constant F.sub.c domains). The drug
blocks the action of the pleiotropic proinflammatory TNF.alpha.
(tumor necrosis factor alpha) by binding to it and preventing it
from signaling the receptors for TNF.alpha. on the surface of
cells. TNF.alpha. is one of the key cytokines that triggers and
sustains the inflammation response.
[0214] Remicade is administered by intravenous infusion, typically
at 6-8 week intervals, and at a clinic or hospital. It cannot be
administered orally, because the digestive system would destroy the
drug unless it is encapsulated in nanoparticles of the present
invention. Infliximab neutralizes the biological activity of
TNF.alpha. by binding with high affinity to the soluble (free
floating in the blood) and transmembrane (located on the outer
membranes of T cells and similar immune cells) forms of TNF.alpha.
and inhibits or prevents the effective binding of TNF.alpha. with
its receptors.
[0215] Remicade and Humira (another TNF antagonist) are in the
subclass of "anti-TNF antibodies" (they are in the form of
naturally occurring antibodies), and are capable of neutralizing
all forms (extracellular, transmembrane, and receptor-bound) of TNF
alpha. Enbrel, a third TNF antagonist, is in a different subclass
(receptor-construct fusion protein), and, because of its modified
form, cannot neutralize receptor-bound TNF.alpha.. Additionally,
the anti-TNF antibodies Humira and Remicade have the capability of
lysing cells involved in the inflammatory process, whereas the
receptor fusion protein apparently lacks this capability. Although
the clinical significance of these differences has not been
absolutely proven, they may account for the discrepancies of these
drugs in both efficacy and side effects.
[0216] Infliximab has high specificity for TNF.alpha., and does not
neutralize TNF beta (TNF.beta., also called lymphotoxin .alpha.), a
related but less inflammatory cytokine that utilizes the same
receptors as TNF.alpha.. Biological activities that are attributed
to TNF.alpha. include: induction of proinflammatory cytokines such
as interleukin (IL-1 and IL-6), enhancement of leukocyte movement
or migration from the blood vessels into the tissues by increasing
the permeability of endothelial layer of blood vessels; and
increasing the release of adhesion molecules. Infliximab prevents
disease in transgenic mice (a special type of mice that are
biologically engineered to produce a human form of TNF.alpha. and
are used to test these drugs for results that might be expected in
humans). These experimental mice develop arthritis as a result of
their production of human TNF.alpha., and when administered after
disease onset, infliximab allows eroded joints to heal.
[0217] Infliximab has a chemical formula
C.sub.6428H.sub.9912N.sub.1694O.sub.1987S.sub.46 and a molecular
mass of 144190.3 g/mol. REMICADE (infliximab) is an advanced
treatment that has been shown to have substantial benefits in
patients with a number of inflammatory disorders involving the
immune system. REMICADE targets specific proteins in the body's
immune system to help control the development of inflammation,
significantly reducing painful symptoms in diseases such as plaque
psoriasis, rheumatoid arthritis, psoriatic arthritis, adult Crohn's
disease, pediatric Crohn's disease, ulcerative colitis, and
ankylosing spondylitis.
[0218] REMICADE is a type of protein that recognizes, attaches to,
and blocks the action of a substance in the body called tumor
necrosis factor (TNF). TNF is made by certain blood cells in the
body. REMICADE will not cure plaque psoriasis, rheumatoid
arthritis, psoriatic arthritis, adult Crohn's disease, pediatric
Crohn's disease, ulcerative colitis, and ankylosing spondylitis,
but blocking TNF may reduce the inflammation caused in the
body.
[0219] Some aspects of the invention relate to a method of reducing
inflammatory response caused by tumor necrosis factor in an animal
subject, the method comprising orally administering nanoparticles
composed of a TNF inhibitor, chitosan, and a core substrate of
poly(glutamic acid) or heparin. In one embodiment, the inhibitor
can be a monoclonal antibody such as infliximab (Remicade) or
adalimumab (Humira), or a circulating receptor fusion protein such
as etanercept (Enbrel). In one embodiment, a TNF inhibitor
nanoparticle formulation consisting of at least one inhibitor
selected from the group consisting of infliximab, adalimumab and
etanercept, chitosan, and one core negatively charged substrate of
poly(glutamic acid) or heparin.
[0220] Nanoparticles Loaded with Bacteriophage
[0221] A bacteriophage is any one of a number of viruses that
infect bacteria. The term is commonly used in its shortened form,
phage. Typically, bacteriophages consist of an outer protein hull
enclosing genetic material. The genetic material can be ssRNA,
dsRNA, ssDNA, or dsDNA (`ss-` or `ds-` prefix denotes single strand
or double strand) between 5 and 500 kilo nucleotides long with
either circular or linear arrangement. Bacteriophages are much
smaller than the bacteria they destroy--usually between 20 and 200
nm in size.
[0222] Phages are estimated to be the most widely distributed and
diverse entities in the biosphere. Phages are ubiquitous and can be
found in all reservoirs populated by bacterial hosts, such as soil
or the intestines of animals. One of the densest natural sources
for phages and other viruses is sea water, where up to
9.times.10.sup.8 virions per milliliter have been found in
microbial mats at the surface, and up to 70% of marine bacteria may
be infected by phages. They have been used for over 60 years as an
alternative to antibiotics in the former Soviet Union and Eastern
Europe. They are seen as a possible therapy against multi drug
resistant strains of many bacteria.
[0223] To enter a host cell, bacteriophages attach to specific
receptors on the surface of bacteria, including
lipopolysaccharides, teichoic acids, proteins, or even flagella.
This specificity means that a bacteriophage can only infect certain
bacteria bearing receptors that they can bind to, which in turn
determines the phage's host range. As phage virions do not move
independently, they must rely on random encounters with the right
receptors when in a solution (blood, lymphatic circulation,
irrigation, soil water etc.).
[0224] Complex bacteriophages use a syringe-like motion to inject
their genetic material into the cell. After making contact with the
appropriate receptor, the tail fibers bring the base plate closer
to the surface of the cell. Once attached completely, the tail
contracts, possibly with the help of ATP present in the tail,
injecting genetic material through the bacterial membrane.
[0225] Within minutes, bacterial ribosomes start translating viral
mRNA into protein. For RNA-based phages, RNA replicase is
synthesized early in the process. Proteins modify the bacterial RNA
polymerase so that it preferentially transcribes viral mRNA. The
host's normal synthesis of proteins and nucleic acids is disrupted,
and it is forced to manufacture viral products instead. These
products go on to become part of new virions within the cell,
helper proteins which help assemble the new virions, or proteins
involved in cell lysis.
[0226] Phages may be released via cell lysis, by extrusion, or, in
a few cases, by budding. Lysis, by tailed phages, is achieved by an
enzyme called endolysin which attacks and breaks down the cell wall
peptidoglycan. An altogether different phage type, the filamentous
phages, causes the host cell to continually secrete new virus
particles. Released virions are described as free and unless
defective are capable of infecting a new bacterium. Budding is
associated with certain Mycoplasma phages. In contrast to virion
release, phages displaying a lysogenic cycle do not kill the host
but rather become long-term residents as prophage.
[0227] In August, 2006 the United States Food and Drug
Administration (FDA) approved using bacteriophages on cheese to
kill the Listeria monocytogenes bacteria, giving them GRAS status
(Generally Recognized As Safe). In July 2007, the same
bacteriophages were approved for use on all food products.
[0228] Some aspects of the invention relate to a method of
mitigating bacteria in an animal subject, the method comprising
orally administering nanoparticles composed of at least one
bacteriophage, chitosan, and a core substrate of poly(glutamic
acid) or heparin. One aspect of the invention relates to a
bactericide nanoparticle formulation comprising at least one
bacteriophage, chitosan, and a core substrate of poly(glutamic
acid) or heparin. In one embodiment, a bactericide nanoparticle
formulation consisting of at least one bacteriophage, chitosan, and
one core negatively charged substrate of poly(glutamic acid) or
heparin. In a further embodiment, the nanoparticles are
encapsulated in capsules, wherein the capsules may be treated with
an enteric coating polymer. In one embodiment, the capsules further
comprise a permeation enhancer, wherein the permeation enhancer is
selected from the group consisting of Ca.sup.2+ chelators, bile
salts, anionic surfactants, medium-chain fatty acids, phosphate
esters, chitosan, and chitosan derivatives. In another embodiment,
the capsule may contain solubilizer such as GRAS or other
pharmacopoeial excipients.
Example No. 21
Nanoparticles Loaded with Pemetrexed
[0229] Pemetrexed (brand name Alimta.RTM.) is a chemotherapy drug
manufactured and marketed by Eli Lilly and Company. Its indications
are the treatment of pleural mesothelioma as well as non-small cell
lung cancer. Pemetrexed has a systematic (IUPAC) name
2-[4-[2-(4-amino-2-oxo-3,5,7-triazabicyclo[4.3.0]nona-3,8,10-trien-9-yl)e-
thyl]benzoyl]aminopentanedioic acid, a chemical formula
C.sub.20H.sub.21N.sub.5O.sub.6 and a molecular mass of 427.411
g/mol. Pemetrexed is chemically similar to folic acid and is in the
class of chemotherapy drugs called folate antimetabolites. It works
by inhibiting three enzymes used in purine and pyrimidine
synthesis--thymidylate synthase (TS), dihydrofolate reductase
(DHFR), and glycinamide ribonucleotide formyltransferase (GARFT).
By inhibiting the formation of precursor purine and pyrimidine
nucleotides, pemetrexed prevents the formation of DNA and RNA,
which are required for the growth and survival of both normal cells
and cancer cells. In February 2004, the Food and Drug
Administration approved pemetrexed for treatment of malignant
pleural mesothelioma, a type of tumor of the lining of the lung, in
combination with cisplatin. In September 2008, the FDA granted
approval as a first-line treatment, in combination with cisplatin,
against of locally-advanced and metastatic non-small cell lung
cancer, or NSCLC, in patients with non-squamous histology. Trials
are currently testing it against esophagus and other cancers.
[0230] In one exemplary preparation, nanoparticles were obtained
upon addition of a mixture of .gamma.-PGA plus pemetrexed aqueous
solution (2 ml), using a pipette (0.5-5 ml, PLASTIBRAND.RTM.,
BrandTech Scientific Inc., Germany), into a low-MW CS aqueous
solution (pH 6.0, 10 ml) at concentrations higher than 0.10% by w/v
under magnetic stirring at room temperature to ensure positive
surface charge. Nanoparticles were collected by ultracentrifugation
at 38,000 rpm for 1 hour. Pemetrexed is wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water as
the solution products, further encapsulated in capsules/enteric
capsules or further treated with lyophilization freeze-dried
process. The nanoparticles thus obtained via the simple and mild
ionic-gelation method described herein show typical characteristics
in a spheroidal configuration with a particle size of between about
50 to 400 nm, a positive surface charge and a narrow polydispersity
index.
[0231] Epirubicin is an anthracycline drug used for chemotherapy.
It is marketed by Pfizer under the trade name Ellence in the U.S.
and Pharmorubicin or Epirubicin elsewhere. Similarly to other
anthracyclines, epirubicin acts by intercalating DNA strands.
Intercalation results in complex formation that inhibits DNA and
RNA synthesis. It also triggers DNA cleavage by topoisomerase
resulting in mechanisms that lead to cell death. Binding to cell
membranes and plasma proteins may be involved in the compound's
cytotoxic effects. Epirubicin also generates free radicals that
cause cell and DNA damage. Epirubicin is favored over doxorubicin,
the most popular anthracycline, in some chemotherapy regimens as it
appears to cause fewer side-effects. Epirubicin has a different
spatial orientation of the hydroxyl group at the 4' carbon of the
sugar, which may account for its faster elimination and reduced
toxicity. Epirubicin is primarily used against breast and ovarian
cancer, gastric cancer, lung cancer, and lymphomas. Its systematic
(IUPAC) name is
10-(4-amino-5-hydroxy-6-methyl-oxan-2-yl)oxy-6,8,11-trihydroxy-8-(2-hydro-
xyacetyl)-1-methoxy-9,10-dihydro-7H-tetracene-5,12-dione. The
chemical formula is C.sub.27H.sub.29NO.sub.11 with a molecular mass
543.519 g/mol.
[0232] Irinotecan is a chemotherapy agent that is a topoisomerase 1
inhibitor. Chemically, it is a semisynthetic analogue of the
natural alkaloid camptothecin. Its main use is in colon cancer,
particularly in combination with other chemotherapy agents.
Irinotecan was first introduced in Japan by the Pharmaceutical arm
of Yakult Honsha as Campto. In 1994, it received accelerated FDA
approval in the United States, where it is now marketed by Pfizer
as Camptosar. It is also known as CPT-11. Its systematic (IUPAC)
name is
(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',':-
6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'bipiperidine]-1'-carboxylate.
It has a chemical formula C.sub.33H.sub.38N.sub.4O.sub.6 with a
molecular mass 677.185 g/mol (hydrochloride). Irinotecan is
activated by hydrolysis to SN-38, an inhibitor of topoisomerase I.
This is then inactivated by glucuronidation by uridine diphosphate
glucoronosyltransferase 1A1 (UGT1A1). The inhibition of
topoisomerase I by the active metabolite SN-38 eventually leads to
inhibition of both DNA replication and transcription.
Example No. 22
Nanoparticles Loaded with Gemcitabine
[0233] Gemcitabine is a nucleoside analog used as chemotherapy. It
is marketed as Gemzar.RTM. by Eli Lilly and Company. Gemcitabine
has a systematic (IUPAC) name as
4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]--
1H-pyrimidin-2-one. Gemcitabine has a chemical formula
C.sub.9H.sub.11F.sub.2N.sub.3O.sub.4 and a molecular mass of
263.198 g/mol. Gemcitabine is used in various carcinomas: non-small
cell lung cancer, pancreatic cancer, bladder cancer and breast
cancer. It is being investigated for use in oesophageal cancer, and
is used experimentally in lymphomas and various other tumor types.
Gemcitabine represents an advance in pancreatic cancer care. It is
also not as debilitating as other forms of chemotherapy. A study
reported in the Journal of the American Medical Association
suggested that gemcitabine shows benefit in patients with
pancreatic cancer who were considered to have successful tumor
resections. GemCarbo chemotherapy (consisting of gemcitabine, as
known as Gemzar and Carboplatin, which are both colourless fluids)
is used to treat several different types of cancer, but most
commonly lung cancer.
[0234] In one exemplary preparation, nanoparticles were obtained
upon addition of a mixture of .gamma.-PGA and gemcitabine aqueous
solution (2 ml), using a pipette (0.5-5 ml, PLASTIBRAND.RTM.,
BrandTech Scientific Inc., Germany), into a low-MW CS aqueous
solution (pH 6.0, 10 ml) at concentrations higher than 0.10% by w/v
under magnetic stirring at room temperature to ensure positive
surface charge. Nanoparticles were collected by ultracentrifugation
at 38,000 rpm for 1 hour. Gemcitabine is wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water as
the solution products, further encapsulated in capsules/enteric
capsules or treated with lyophilization freeze-dried process. The
nanoparticles thus obtained via the simple and mild ionic-gelation
method described herein show typical characteristics in a
spheroidal configuration with a particle size of between about 50
to 400 nm, a positive surface charge, and a narrow polydispersity
index.
Example No. 23
Nanoparticles Loaded with Drotrecogin Alfa (Activated)
[0235] Drotrecogin alfa (activated) (Xigrist, marketed by Eli Lilly
and Company) is a recombinant form of human activated protein C
that has anti-thrombotic, anti-inflammatory, and profibrinolytic
properties. Drotrecogin alpha (activated) belongs to the class of
serine proteases. It is used mainly in intensive care medicine as a
treatment for severe sepsis. Drotrecogin alpha (activated) has a
systematic (IUPAC) name as Activated human protein C; it has a
chemical formula C.sub.1786H.sub.2779N.sub.509O.sub.519S.sub.29 and
a molecular mass of 55000 g/mol. The specific mechanism by which
drotrecogin exerts its effect on survival in patients with severe
sepsis is not completely understood. In vitro data suggest that
activated protein C exerts an antithrombotic effect by inhibiting
factors Va and VIIIa, and that it has indirect profibrinolytic
activity by inhibiting plasminogen activator inhibitor-1 (PAI-1).
In vitro data also suggest that activated protein C may exert an
anti-inflammatory effect by inhibiting tumor necrosis factor
production, by blocking leukocyte adhesion to selectins, and by
limiting the thrombin-induced inflammatory responses within the
microvascular endothelium. If the i.v. dosage guidelines are
followed, the drug reaches peak plasma levels after two hours and
is completely cleared from plasma two hours after the termination
of the infusion period. Endogenous plasma protease inhibitors
deactivate drotrecogin. Therefore, no dosage adjustment is needed
in elderly patients, or in patients with renal or hepatic
dysfunction. Drotrecogin is indicated for the reduction of
mortality in adult patients with severe sepsis (sepsis associated
with acute organ dysfunction) who have a high risk of death.
[0236] In one exemplary preparation, nanoparticles were obtained
upon addition of a mixture of .gamma.-PGA and drotrecogin alpha
(activated) aqueous solution (2 ml), using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
low-MW CS aqueous solution (pH 6.0, 10 ml) at concentrations higher
than 0.10% by w/v under magnetic stirring at room temperature to
ensure positive surface charge. Nanoparticles were collected by
ultracentrifugation at 38,000 rpm for 1 hour. Drotrecogin alpha
(activated) is wholly or substantially totally encapsulated within
the nanoparticles. Supernatants were discarded and nanoparticles
were resuspended in deionized water as the solution products. Then
they were either encapsulated in capsules/enteric capsules or
treated with lyophilization freeze-dried process. The nanoparticles
thus obtained via the simple and mild ionic-gelation method
described herein show typical characteristics in a spheroidal
configuration with a particle size of between about 50 to 400 nm, a
positive surface charge and a narrow polydispersity index.
[0237] Factor IX (or Christmas factor) is one of the serine
proteases of the coagulation system; it belongs to peptidase family
S1. Deficiency of this protein causes hemophilia B. Factor IX is
inactive unless activated by factor XIa (of the contact pathway) or
factor VIIa (of the tissue factor pathway). When activated into
factor IXa, it acts by hydrolysing one arginine-isoleucine bond in
factor X to form factor Xa. It requires calcium, membrane
phospholipids, and factor VIII as cofactors to do so. Factor IX
complex is currently via injectable routes. Its generic name is
Factor IX, human recombinant-injection. In use, Factor IX is a part
of blood needed for clotting which stops bleeding. Persons with low
Factor levels are at risk for bleeding. This medication is used to
prevent or control bleeding episodes in persons with low Factor
levels (hemophilia, Christmas disease). It is also used to reverse
the effects of warfarin blood thinner. Factor IX has been
incorporated in the nanoparticle formulation via the simple and
mild ionic-gelation method described herein, the nanoparticles thus
obtained show typical characteristics in a spheroidal configuration
with a particle size of between about 50 to 400 nm, a positive
surface charge and a narrow polydispersity index.
Example No. 24
Nanoparticles Loaded with Humatrope
[0238] Humatrope.RTM. (somatotropin or somatropin) is a polypeptide
hormone of rDNA origin. Manufactured by Eli Lilly and Company, it
is used to stimulate linear growth in pediatric patients who lack
adequate normal human growth hormone. It has 191 amino acid
residues and a molecular weight of 22,125 daltons. Its amino acid
sequence is identical to that of human growth hormone of pituitary
origin (anterior lobe). Humatrope is synthesized in a strain of E.
coli modified by the addition of a gene for human growth hormone.
Other human growth hormone produced from rDNAs include;
Omnitrope.RTM. (Sandoz), Nutropin, Norditropin, Genotropin.RTM.
(Pfizer).
[0239] In one exemplary preparation, nanoparticles were obtained
upon addition of a mixture of .gamma.-PGA and Humatrope aqueous
solution (2 ml), using a pipette (0.5-5 ml, PLASTIBRAND.RTM.,
BrandTech Scientific Inc., Germany), into a low-MW CS aqueous
solution (pH 6.0, 10 ml) at concentrations higher than 0.10% by w/v
under magnetic stirring at room temperature to ensure positive
surface charge. Nanoparticles were collected by ultracentrifugation
at 38,000 rpm for 1 hour. Humatrope is wholly or substantially
totally encapsulated within the nanoparticles. Supernatants were
discarded and nanoparticles were resuspended in deionized water as
the solution products, further encapsulated in capsules/enteric
capsules or treated with lyophilization freeze-dried process. The
nanoparticles thus obtained via the simple and mild ionic-gelation
method described herein show typical characteristics in a
spheroidal configuration with a particle size of between about 50
to 400 nm, a positive surface charge, and a narrow polydispersity
index.
[0240] The major isoform of the human growth hormone (GH) is a
protein of 191 amino acids and a molecular weight of 22124 daltons.
The structure includes four helices necessary for functional
interaction with the GH receptor. GH is structurally and apparently
evolutionarily homologous to prolactin and chorionic
somatomammotropin. Despite marked structural similarities between
growth hormone from different species, only human and primate
growth hormones have significant effects in humans. Effects of
growth hormone on the tissues of the body can generally be
described as anabolic (building up). Like most other protein
hormones, GH acts by interacting with a specific receptor on the
surface of cells. In one aspect, GH stimulating the increase in
height in childhood is the most widely known effect of GH, and
appears to be caused by at least two mechanisms. In another aspect,
because polypeptide hormones are not fat soluble, they cannot
penetrate sarcolemma. Thus, GH exerts some of its effects by
binding to receptors on target cells, where it activates a
secondary messenger. Through this mechanism, GH directly stimulates
division and multiplication of chondrocytes of cartilage. These are
the primary cells in the growing ends (epiphyses) of children's
long bones.
[0241] GH also stimulates production of insulin-like growth factor
1 (IGF-1, formerly known as somatomedin C), a hormone homologous to
proinsulin. The liver is a major target organ of GH for this
process, and is the principal site of IGF-1 production. IGF-1 has
growth-stimulating effects on a wide variety of tissues. Additional
IGF-1 is generated within target tissues, making it apparently both
an endocrine and an autocrine/paracrine hormone. IGF-1 also has
stimulatory effects on osteoblast and chondrocyte activity to
promote bone growth. In addition to increasing height in children
and adolescents, growth hormone has many other effects on the body,
such as: increasing calcium retention, and strengthening and
increasing the mineralization of bone; increasing muscle mass
through sarcomere hyperplasia; promoting lipolysis; increasing
protein synthesis; stimulating the growth of all internal organs
excluding the brain; playing a role in fuel homeostasis; reducing
liver uptake of glucose; promoting gluconeogenesis in the liver;
contributing to the maintenance and function of pancreatic islets;
and stimulating the immune system.
[0242] Pegylation
[0243] Pegylation is the process of covalent attachment of
polyethylene glycol polymer chains to another molecule, normally a
drug or therapeutic protein or bioactive agent (i.e., pegylated
bioactive agent) or chitosan (i.e., PEG-CS). Pegylation is
routinely achieved by incubation of a reactive derivative of PEG
with the target macromolecule. The covalent attachment of PEG to a
drug or therapeutic protein can "mask" the agent from the host's
immune system (reduced immunogenicity and antigenicity), increase
the hydrodynamic size (size in solution) of the agent which
prolongs its circulatory time by reducing renal clearance.
Pegylation can also provide water solubility to hydrophobic drugs
and proteins, molecules most typically peptides, proteins, and
antibody fragments that can help to meet the challenges of
improving the safety and efficiency of many therapeutics. It
produces alterations in the physiochemical properties including
changes in conformation, electrostatic binding, hydrophobicity etc.
These physical and chemical changes increase systemic retention of
the therapeutic agent. Also, it can influence the binding affinity
of the therapeutic moiety to the cell receptors and can alter the
absorption and distribution patterns.
[0244] Pegylation, by increasing the molecular weight of a
molecule, can impart several significant pharmacological advantages
over the unmodified form, such as: improved molecule (drug)
solubility, reduced dosage frequency, without diminished efficacy
with potentially reduced toxicity, extended circulating life,
increased molecule (drug) stability, and enhanced protection from
proteolytic degradation. Pegylated drugs have the following
commercial advantages such as opportunities for new delivery
formats and dosing regimens and extended patent life of previously
approved drugs.
[0245] The first step of the Pegylation is the suitable
functionalization of the PEG polymer at one or both terminals. PEGs
that are activated at each terminus with the same reactive moiety
are known as "homobifunctional", whereas if the functional groups
present are different, then the PEG derivative is referred as
"heterobifunctional" or "heterofunctional." The chemically active
or activated derivatives of the PEG polymer are prepared to attach
the PEG to the desired molecule. The overall Pegylation processes
used to date for protein conjugation can be broadly classified into
two types, namely a solution phase batch process and an on-column
fed-batch process. The simple and commonly adopted batch process
involves the mixing of reagents together in a suitable buffer
solution, preferably at a temperature between 4 and 6.degree. C.,
followed by the separation and purification of the desired product
using a suitable technique based on its physicochemical properties,
including size exclusion chromatography (SEC), ion exchange
chromatography (IEX), hydrophobic interaction chromatography (HIC)
and membranes or aqueous two phase systems.
[0246] The choice of the suitable functional group for the PEG
derivative is based on the type of available reactive group on the
molecule that will be coupled to the PEG. For proteins, typical
reactive amino acids include lysine, cysteine, histidine, arginine,
aspartic acid, glutamic acid, serine, threonine, and tyrosine. The
N-terminal amino group and the C-terminal carboxylic acid can also
be used as a site-specific site by conjugation with aldehyde
functional polymers.
[0247] The techniques used to form first generation PEG derivatives
are generally reacting the PEG polymer with a group that is
reactive with hydroxyl groups, typically anhydrides, acid
chlorides, chloroformates and carbonates. In the second generation
pegylation chemistry more efficient functional groups such as
aldehyde, esters, amides, etc made available for conjugation. As
applications of pegylation have become more and more advanced and
sophisticated, there has been an increase in need for
heterobifunctional PEGs for conjugation. These heterobifunctional
PEGs are very useful in linking two entities, where a hydrophilic,
flexible and biocompatible spacer is needed. Preferred end groups
for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl
disulfide, amine, carboxylic acids and NHS esters. Third generation
pegylation agents, where the shape of the polymer has been
branched, Y shaped or comb shaped are available which show reduced
viscosity and lack of organ accumulation.
[0248] Some aspects of the invention relate to a pharmaceutical
composition of nanoparticles, the nanoparticles consisting of a
shell portion that is dominated by positively charged chitosan, a
core portion that consists of the positively charged chitosan, one
negatively charged substrate, at least one pegylated bioactive
agent loaded within the nanoparticles, and optionally a zero-charge
compound. In one embodiment, the pegylated bioactive agent is an
anti-diabetic drug that is covalently attached polyethylene glycol
polymer chains. In another embodiment, the pegylated anti-diabetic
drug is selected from the group consisting of insulin, an insulin
analog, GLP-1, a GLP-1 analog, an insulin sensitizer, an insulin
secretagogue, an inhibitor of dipeptidyl peptidase 4, metformin,
alpha-glucosidase inhibitors, amylin analog, sodium-glucose
co-transporter type 2 (SGLT2) inhibitors, benfluorex, tolrestat,
and combinations thereof.
Example No. 25
Nanoparticles Loaded with Anti-Hemophilic Factors
[0249] Hemophilia is a group of hereditary genetic disorders that
impair the body's ability to control blood clotting or coagulation.
The effects of this sex-linked, X chromosome disorder are
manifested almost entirely in males, although the gene for the
disorder is inherited from the mother. Females have two X
chromosomes while males have only one, lacking a `back up` copy for
the defective gene. Females are therefore almost exclusively
carriers of the disorder, and may have inherited it from either
their mother or father. These genetic deficiencies may lower blood
plasma clotting factor levels of coagulation factors needed for a
normal clotting process. When a blood vessel is injured, a
temporary scab does form, but the missing coagulation factors
prevent fibrin formation which is necessary to maintain the blood
clot. Thus, a hemophiliac does not bleed more intensely than a
normal person, but for a much longer amount of time. In severe
hemophiliacs, even a minor injury could result in blood loss
lasting days, weeks, or may never heal completely. The critical
risk here is with normally small injuries, which, due to missing
factor VIII, take extended periods of time to heal. In areas such
as the brain or inside joints, this can be fatal or permanently
debilitating.
[0250] In one exemplary preparation, nanoparticles were obtained
upon addition of a mixture of .gamma.-PGA and SonoSeven (a
recombinant human coagulation Factor VIIa; rFVIIa) aqueous solution
(2 ml), using a pipette (0.5-5 ml, PLASTIBRAND.RTM., BrandTech
Scientific Inc., Germany), into a low-MW CS aqueous solution (pH
6.0, 10 ml) at concentrations higher than 0.10% by w/v under
magnetic stirring at room temperature to ensure positive surface
charge. Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour. rFVIIa is wholly or substantially totally
encapsulated within the nanoparticles. Supernatants were discarded
and nanoparticles were resuspended in deionized water as the
solution products. Then they were either encapsulated in
capsules/enteric capsules or treated with lyophilization
freeze-dried process. The nanoparticles thus obtained via the
simple and mild ionic-gelation method described herein show typical
characteristics in a spheroidal configuration with a particle size
of between about 50 to 400 nm, a positive surface charge, and a
narrow polydispersity index.
[0251] Hemophilia A is an X-linked genetic disorder involving a
lack of functional clotting Factor VIII and represents 90% of
hemophilia cases. Hemophilia B is an X-linked genetic disorder
involving a lack of functional clotting Factor IX. It is less
severe but more uncommon than Hemophilia A. Hemophilia C is an
autosomal recessive genetic disorder involving a lack of functional
clotting Factor XI. Though there is no cure for hemophilia, it can
be conventionally controlled with regular infusions of the
deficient clotting factor, i.e. factor VIII in hemophilia A or
factor IX in hemophilia B. Factor replacement can be either
isolated from human blood serum, recombinant, or a combination of
the two. Some hemophiliacs develop antibodies (inhibitors) against
the replacement factors given to them, so the amount of the factor
has to be increased or non-human replacement products must be
given, such as porcine factor VIII. Inhibitors are a complication
of hemophilia. People with severe Hemophilia A or B is usually
treated by replacing the missing Factor VIII or Factor IX through
infusion. For some people, however, this treatment does not work.
Their bodies react as though the treatment is an invader and their
immune system develops antibodies (inhibitors) which attack and
neutralize the Factor VIII or IX. The neutralized factor is not
able to stop the bleeding.
[0252] In one aspect, Xyntha.TM. (Wyeth) anti-hemophilic factor
(recombinant), plasma/serum-free is indicated for the control and
prevention of bleeding episodes in an animal subject with
hemophilia A (congenital factor VIII deficiency or classic
hemophilia) and for surgical prophylaxis in an animal subject with
hemophilia A. Patients can control bleeding episodes with normal
plasma, concentrates of factor VII, or genetically produced
(recombinant) factor VII. People need frequent treatment during
bleeding episodes because factor VII does not last long. Women can
control menstrual bleeding with oral contraceptives.
[0253] An activated concentrate of factor VII called NovoSeven can
also be used. In one aspect, NovoSeven.RTM. (Novo Nordisk) is a
recombinant human coagulation Factor Vila (rFVIIa), intended for
promoting hemostasis by activating the extrinsic pathway of the
coagulation cascade. NovoSeven is a vitamin k-dependent
glycoprotein consisting of 406 amino acid residues (MW 50 k
Dalton). NovoSeven is structurally similar to human plasma-derived
Factor VIIa. NovoSeven is supplied as a sterile, white lyophilized
powder of rFVIIa in single-use vials. Some other brand names for
the generic anti-hemophilic factor, recombinant--injection may
include: Bioclate (Aventis Behring), Helixate (CSL Behring),
Kogenate (Bayer Healthcare), Recombinate (Baxter Healthcare) Advate
(Baxter Healthcare), Alphanate (Grifols SA), Hemofil-M (Baxter
Healthcare), Humate-P (CLS Behrng), Koate (Talecris
Biotherapeutics), Monarc-M (Baxter Healthcare), Monoclate-P (CSL
Behring), Refacto (Wyeth), and others.
[0254] Idarubicin or 4-demethoxydaunorubicin is an anthracycline
antileukemic drug that is currently combined with cytosine
arabinoside as a first line treatment of acute myeloid leukemia. It
belongs to the family of drugs called antitumor antibiotics. It is
distributed under the trade names Zavedos (UK) and Idamycin
(USA).
[0255] Some aspects of the invention relate to a method of
promoting hemostasis caused by hemophilia in an animal subject, the
method comprising orally administering nanoparticles composed of
anti-hemophilic factor, chitosan, and a core substrate of
poly(glutamic acid) or heparin, wherein a surface of the
nanoparticles is dominated by chitosan. In one embodiment, the oral
nanoparticles comprise chitosan or chitosan derivatives as a
permeation enhancer. In another embodiment, the nanoparticles
further comprise a permeation enhancer.
[0256] Some aspects of the invention relate to a pharmaceutical
composition for treating a subject comprising two or more bioactive
nanoparticles, thus treating the subject by co-administering the
bioactive nanoparticles to the subject, wherein a first bioactive
nanoparticle comprises a shell portion that is dominated by
positively charged chitosan, a core portion that contains
negatively charged substrate, wherein the negatively charged
substrate is at least partially neutralized with a portion of the
positively charged chitosan, and at least a first bioactive agent,
and wherein a second bioactive nanoparticle comprises a shell
portion that is dominated by positively charged chitosan, a core
portion that contains negatively charged substrate, wherein the
negatively charged substrate is at least partially neutralized with
a portion of the positively charged chitosan, and at least a second
bioactive agent.
[0257] In one embodiment, the first and second nanoparticles are
loaded in same capsules. In another embodiment, the first
nanoparticle is loaded in a first capsule and the second
nanoparticle is loaded in a second capsule. In one embodiment, the
capsules are treated with enteric coating. In a further embodiment,
the capsules further comprise at least a solubilizer or
pharmacopoeial excipients. In another embodiment, the capsules
further comprise a permeation enhancer, wherein the permeation
enhancer is selected from the group consisting of Ca.sup.2+
chelators, bile salts, anionic surfactants, medium-chain fatty
acids, phosphate esters, chitosan, and chitosan derivatives. In one
embodiment, the first and second nanoparticles are loaded in
tablets or pills.
[0258] In one embodiment, the first bioactive agent of the
pharmaceutical composition comprises non-insulin anti-diabetic
drug, wherein the non-insulin anti-diabetic drug is selected from
the group consisting of insulin sensitizers, insulin secretagogues,
GLP-1 analogs, and DPP-4 inhibitors, alpha-glucosidase inhibitors,
amylin analog, sodium-glucose co-transporter type 2 (SGLT2)
inhibitors, benfluorex, and tolrestat. In another embodiment, the
second bioactive agent of the pharmaceutical composition comprises
insulin or insulin analogs.
Example No. 26
Nanoparticles Loaded with DTPA
[0259] Some aspects of the invention relate to a pharmaceutical
composition of nanoparticle comprising chitosan, PGA-complexone
conjugate and a bioactive agent. In one embodiment, the
PGA-complexone conjugate may broadly include a conjugate with PGA
derivatives such as .gamma.-PGA, .alpha.-PGA, derivatives of PGA or
salts of PGA, whereas the complexone may cover DTPA (diethylene
triamine pentaacetic acid), EDTA (ethylene diamine tetra acetate),
IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), EGTA
(ethylene glycol tetraacetic acid), BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), DOTA
(1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid), NOTA
(2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid), and the
like. A polyamino carboxylic acid (complexone) is a compound
containing one or more nitrogen atoms connected through carbon
atoms to one or more carboxyl groups.
[0260] Diethylene triamine pentaacetic acid (DTPA) is a polyamino
carboxylic acid consisting of a diethylenetriamine backbone
modified with five carboxymethyl groups. The molecule can be viewed
as an expanded version of EDTA. DTPA is used as its conjugate base,
often undefined, which has a high affinity for metal cations. In
example, upon complexation to lanthanide and actinide ions, DTPA
exists as the pentaanionic form, i.e. all five carboxylic acid
groups are deprotonated. DTPA has a molecular formula of
C.sub.14H.sub.23N.sub.3O.sub.10 with molar mass 393.358 g/mole. The
chemical formula for DTPA and EGTA are shown below as:
##STR00008##
[0261] Currently, DTPA is approved by the U.S. Food and Drug
Administration (FDA) for chelation of three radioactive materials:
plutonium, americium, and curium. DTPA is the parent acid of an
octadentate ligand, diethylene triamine pentaacetate. In some
situations, all five acetate arms are not attached to the metal
ion. In one aspect of the present invention, DTPA has been
conjugated to .gamma.-PGA through hexanediamine
((.gamma.-PGA)-DTPA) as illustrated below:
##STR00009##
[0262] In one aspect of the invention, (.gamma.-PGA)-DTPA is one
species of the PGA-complexone conjugates used in the current
pharmaceutical composition of nanoparticles. The overall degree of
substitution of DTPA in (.gamma.-PGA)-DTPA conjugate is generally
in the range of about 1-70%, preferably in the range of about
5-40%, and most preferably in the range of about 10-30%. DTPA does
not build up in the body or cause long-term health effects.
[0263] Nanoparticles comprising chitosan, PGA-complexone conjugates
and at least one bioactive agent using the simple and mild
ionic-gelation process described herein has demonstrated the
desired paracellular transport efficacy with TEER measurements in
the Caco-2 cell cultures model as described in Example No. 4.
Example No. 27
Enzyme Inhibition Study with (.gamma.-PGA)-DTPA Conjugate
[0264] Brush border membrane bounded enzymes were used to simulate
a contacting membrane at the bottom of a donor compartment, wherein
the insulin-loaded medium (Krebs-Ringer buffer) in the donor
compartment was used as the starting material at time zero. Three
elements were used in this enzyme inhibition study to assess the
enzymatic degradation of insulin versus time by brush border
membrane bounded enzymes. They were (a) insulin 1 mg/ml as control;
(b) DTPA 5 mg/ml; and (c) (.gamma.-PGA)-DTPA 5 mg/ml. As shown in
FIG. 20, both DTPA and (.gamma.-PGA)-DTPA substantially protect or
maintain the insulin activity or viable content over the
experimental duration up to 2 hours. More particularly, the enzyme
inhibition index of (.gamma.-PGA)-DTPA is consistently higher than
that of DTPA alone at all sample measuring points of time. This is
evident that (.gamma.-PGA)-DTPA conjugate compound is a desired
compound for mitigating the enzymatic effect in the
gastrointestinal tract when co-administered with a peptide or
protein drug orally. Some aspects of the present invention relate
to a method of delivering a peptide or protein drug to an animal
subject with enhanced enzymatic inhibition property (index), the
method comprising co-administering a PGA-complexone conjugate and
the drug to the animal subject orally.
[0265] Some aspects of the invention provide a method of enhancing
enzymatic resistance of a bioactive agent in oral administration by
co-administering the bioactive agent and PGA-complexone to an
animal subject.
[0266] Enzyme Inhibition in Gastrointestinal Tract
[0267] Most peptide drugs are susceptible to degradation by
digestive enzymes presented in the gastrointestinal fluid, such as
trypsin, chymotrypsin, elastase, carboxypeptidases and
aminopeptidases, as well as by di-peptidases and tri-peptidases. In
addition, some peptides are degraded by specific enzymes, such as
the insulin-degrading enzyme present in the cytosol. To overcome
the enzymatic barrier in the gastrointestinal tract (GIT), peroral
peptide drugs have been co-administered with protease inhibitors,
such as Bowman-Birk inhibitor from soybeans, ovomucoid and
aprotinin bacitracins. Some aspects of the present invention relate
to co-administering a peptide/protein drug and an enzyme inhibitive
compound that is non-enzyme specific in oral drug delivery. In one
embodiment, the PGA-complexone (for example, (.gamma.-PGA)-DTPA
conjugate) is in a class of enzyme inhibitive compounds that are
non-enzyme specific.
[0268] Intestinal Applications of CS-NPs in Inflammatory Bowel
Diseases
[0269] In medicine, inflammatory bowel disease (IBD) is a group of
inflammatory conditions of the colon and small intestine. The major
types of IBD are Crohn's disease (autoimmune origin), and
ulcerative colitis. Accounting for far fewer cases are other forms
of IBD, which are not always classified as typical IBD: collagenous
colitis, lymphocytic colitis, ischemic colitis, diversion colitis,
Behcet's disease and indeterminate colitis. This targeted delivery
to inflamed tissue in IBDs is based on the principle of
pH-responsive chitosan/.gamma.-PGA NPS wherein the .gamma.-PGA may
be a conjugate version such as (.gamma.-PGA)-DTPA,
(.gamma.-PGA)-EGTA, (.gamma.-PGA)-EDTA or the like with a
complexion species.
[0270] Optimal treatment of inflammatory bowel disease depends on
what form it consists of. For example, mesalazine is more useful in
ulcerative colitis than in Crohn's disease. Generally, depending on
the level of severity, IBD may require immunosuppression to control
the symptom, such as prednisone, TNF inhibition, azathioprine
(Imuran), methotrexate or 6-mercaptopurine. More commonly,
treatment of IBD requires a form of mesalazine. Often, steroids are
used to control disease flares and were once acceptable as a
maintenance drug. In use for several years in Crohn's disease
patients and recently in patients with ulcerative colitis,
biologicals have been used such as TNF inhibitors. Severe cases may
require surgery, such as bowel resection, strictureplasty or a
temporary or permanent colostomy or ileostomy. Alternative medicine
treatments for bowel disease exist in various forms, however such
methods concentrate on controlling underlying pathology in order to
avoid prolonged steroidal exposure or surgical excisement.
[0271] Usually the treatment is started by administering drugs with
high anti-inflammatory effects, such as prednisone. Once the
inflammation is successfully controlled, the patient is usually
switched to a lighter drug to keep the disease in remission, such
as Asacol, a mesalamine. If unsuccessful, a combination of the
aforementioned immunosuppression drugs with a mesalamine (which may
also have an anti-inflammatory effect) may or may not be
administered, depending on the patient. Histoplasma produces toxins
that cause intestinal disease called histoplasmosis that is a
"serious consideration" in an immunocompromised patient with signs
and symptoms of IBD. Antifungal drugs such as nystatin (a broad
spectrum gut antifungal) and either itraconazole (Sporanox) or
fluconazole (Diflucan) have been suggested as a treatment for IBD
disorders such as Crohn's disease and ulcerative colitis that all
share the same symptoms such as diarrhea, weight loss, fever, and
abdominal pain.
[0272] New evidence suggests that patients with IBD may have an
elevated risk of endothelial dysfunction and coronary artery
disease. The extra-intestinal complication of Crohn's disease and
ulcerative colitis may include iritis, uveitis, primary sclerosing
cholangitis, ankylosing spondylitis, pyoderma gangrenosum, ethryema
nodosum, and others.
[0273] More specifically, the pH in inflamed tissue is about pH 5.5
or less. (NPs have higher zeta potential at this pH) whereas
Crohn's disease is generally located in ileum or colon where normal
pH is 7.0-8.0. Chitosan-shelled NPs can adhere more strongly to the
inflamed tissue as compared to the normal tissue because its pH
sensitivity. More targets in inflamed tissue can be approached by
chitosan-shelled NPs for better specificity of delivery system.
Some aspects of the invention relate to method of treating an
inflammatory bowel disease of an animal subject, the method
comprising administering bioactive nanoparticles to the animal
subject orally, wherein the bioactive nanoparticles consist of at
least one anti-inflammatory agent, positively charged chitosan,
optionally a zero-charge substance or bioactive agent, and a
negatively charged substrate, wherein a surface of the
nanoparticles is dominated by the positively charged chitosan.
[0274] Blood-Brain Barrier and Tight Junctions
[0275] The blood-brain barrier (BBB) is a membrane structure in the
central nervous system (CNS) that restricts the passage of various
chemical substances and microscopic objects (e.g. bacteria) between
the bloodstream and the neural tissue itself, while still allowing
the passage of substances essential to metabolic function. This
"barrier" results from the selectivity of the tight junctions
between endothelial cells in CNS vessels that restricts the passage
of solutes. At the interface between blood and brain, endothelial
cells and associated astrocytes are joined together by structures
called tight junctions. The tight junction is composed of smaller
subunits, frequently dimers that are transmembrane proteins such as
occludin, claudins, junctional adhesion molecule (JAM), ESAM and
others. Each of these transmembrane proteins is anchored into the
endothelial cells by another protein complex that includes ZO-1 and
associated proteins. The blood-brain barrier is composed of
high-density cells restricting passage of substances from the
bloodstream much more than endothelial cells in capillaries
elsewhere in the body.
[0276] Some diseases associated with the blood-brain barrier may
include Meningitis, which is inflammation of the membranes which
surround the brain and spinal cord (these membranes are also known
as meninges). Meningitis is most commonly caused by infections with
various pathogens, examples of which are Staphylococcus aureus and
Haemophilus influenza. When the meninges are inflamed, the
blood-brain barrier may be disrupted. This disruption may increase
the penetration of various substances (including antibiotics) into
the brain. Some aspects of the invention relate to a method for
delivering therapeutic nanoparticles of the present invention
incorporating meningitis antagonist or anti-inflammatory drugs as a
bioactive agent to the tight junction of a brain-blood barrier site
for treatment of meningitis.
[0277] Another disease associated with brain-blood barrier may be
Epilepsy, which is a common neurological disease characterized by
frequent and often untreatable seizures. Several clinical and
experimental data have implicated failure of blood-brain barrier
function in triggering chronic or acute seizures. These findings
have shown that acute seizures are a predictable consequence of
disruption of the BBB by either artificial or inflammatory
mechanisms. In addition, expression of drug resistance molecules
and transporters at the BBB are a significant mechanism of
resistance to commonly used anti-epileptic drugs. Some aspects of
the invention relate to a method for delivering therapeutic
nanoparticles of the present invention incorporating anti-epileptic
drugs or anti-inflammatory drugs/medicine as a bioactive agent to
the tight junction of a brain-blood barrier site for treatment of
epilepsy.
[0278] Another disease associated with brain-blood barrier is
Multiple Sclerosis (MS), which is considered an auto-immune
disorder in which the immune system attacks the myelin protecting
the nerves in the central nervous system. Normally, a person's
nervous system would be inaccessible for the white blood cells due
to the blood-brain barrier. However, it has been shown using MRI
(Magnetic Resonance Imaging) that, when a person is undergoing an
MS "attack," the blood-brain barrier has broken down in a section
of the brain or spinal cord, allowing white blood cells called T
lymphocytes to cross over and destroy the myelin. It has been
suggested that, rather than being a disease of the immune system,
MS is a disease of the blood-brain barrier. It is believed that
oxidative stress plays an important role into the breakdown of the
barrier; anti-oxidants such as lipoic acid may be able to stabilize
a weakening blood-brain barrier. Some aspects of the invention
relate to a method for delivering therapeutic nanoparticles of the
present invention incorporating anti-oxidants or anti-inflammatory
medicine as a bioactive agent to the tight junction of a
brain-blood barrier site for treatment of multiple sclerosis.
[0279] One disease associated with brain-blood barrier is
Neuromyelitis optica, also known as Devic's disease, which is
similar to and often confused with multiple sclerosis. Patients
with neuromyelitis optica have high levels of antibodies against a
protein called aquaporin-4. Some aspects of the invention relate to
a method for delivering therapeutic nanoparticles of the present
invention incorporating anti-neuromyelitis optica drugs or
anti-inflammatory medicine as a bioactive agent to the tight
junction of a brain-blood barrier site for treatment of Devic's
disease.
[0280] One disease associated with brain-blood barrier is
Late-stage neurological trypanosomiasis, or sleeping sickness,
which is a condition in which trypanosoma protozoa are found in
brain tissue. It is not yet known how the parasites infect the
brain from the blood, but it is suspected that they cross through
the choroid plexus, a circumventricular organ. Some aspects of the
invention relate to a method for delivering therapeutic
nanoparticles of the present invention incorporating
anti-neurological trypanosomiasis drugs or anti-inflammatory
medicine as a bioactive agent to the tight junction of a
brain-blood barrier site for treatment of Late-stage neurological
trypanosomiasis.
[0281] One disease associated with brain-blood barrier is
Progressive multifocal leukoencephalopathy (PML), which is a
demyelinating disease of the central nervous system caused by
reactivation of a latent papovavirus (the JC polyomavirus)
infection, that can cross the BBB. Some aspects of the invention
relate to a method for delivering therapeutic nanoparticles of the
present invention incorporating anti-virus (such as papovarus)
drugs as a bioactive agent to the tight junction of a brain-blood
barrier site for treatment of PML or infection.
[0282] One disease associated with brain-blood barrier is HIV
Encephalitis. It is believed that HIV can cross the blood-brain
barrier inside circulating monocytes in the bloodstream ("Trojan
horse theory"). Once inside, these monocytes become activated and
are transformed into macrophages. Activated monocytes release
virions into the brain tissue proximate to brain microvessels.
These viral particles likely attract the attention of sentinel
brain microglia and initiate an inflammatory cascade that may cause
tissue damage to the BBB. This inflammation is HIV encephalitis
(HIVE). Instances of HIVE probably occur throughout the course of
AIDS and is a precursor for HIV-associated dementia (HAD). Some
aspects of the invention relate to a method for delivering
therapeutic nanoparticles of the present invention incorporating
anti-HIV drugs or anti-inflammatory medicine as a bioactive agent
to the tight junction of a brain-blood barrier site for treatment
of HIV.
[0283] Among all diseases associated with blood-brain barrier, the
most critical is Alzheimer's Disease (AD). New evidence indicates
that disruption of the blood-brain barrier in AD patients allows
blood plasma containing amyloid beta (A.beta.) to enter the brain
where the A.beta. adheres preferentially to the surface of
astrocytes. These findings have led to the hypotheses that (i)
breakdown of the blood-brain barrier allows access of
neuron-binding autoantibodies and soluble exogenous A.beta.42 to
brain neurons and (ii) binding of these autoantibodies to neurons
triggers and/or facilitates the internalization and accumulation of
cell surface-bound A.beta.42 in vulnerable neurons through their
natural tendency to clear surface-hound autoantibodies via
endocytosis. Eventually the astrocyte is overwhelmed, dies,
ruptures, and disintegrates, leaving behind the insoluble A.beta.42
plaque. Thus, in some patients, Alzheimer's disease may be caused
(or more likely, aggravated) by a breakdown in the blood-brain
barrier. Some aspects of the invention relate to a method for
delivering therapeutic nanoparticles of the present invention
incorporating anti-Alzheimer's drugs (i.e., Alzheimer's antagonist)
or anti-inflammatory medicine as a bioactive agent to the tight
junction of a brain-blood barrier site for treatment of AD. In one
embodiment, the at least one bioactive agent is an antagonist for
Alzheimer's disease or is for treating Alzheimer's disease selected
from the group consisting of memantine hydrochloride, donepezil
hydrochloride, rivastigmine tartrate, galantamine hydrochloride,
and tacrine hydrochloride.
Example No. 28
Bioactive Nanoparticles Delivery Toward Tight Junctions
[0284] One possible route of a drug administered by the nasal
pathway is to enter the olfactory mucosa, followed by entering the
brain tissue via cerebrospinal fluid (CSF). The mammalian nasal
cavity is lined with three types of epithelia: squamous,
respiratory and olfactory. The main part of the nasal cavity is
covered by a typical airway epithelium. CSF is secreted at the four
choroids plexi, located in the lateral and third and fourth
ventricles. CSF is an isotonic aqueous solution with the
concentrations of the major solutes practically identical to those
found in the plasma, except for K.sup.+ and Ca.sup.2+. Paracellular
passage, followed by transport through the olfactory perineural
space, that may be continuous with a subarachnoid extension that
surrounds the olfactory nerve as it penetrates the cribriform
plate, has been suggested (Arch Otolaryngology 1985; 105:180-184).
Therefore, substances may enter the brain after paracellular
passage by flushing with CSF re-entering again into the brain
extracellular space at the cribriform plate.
[0285] The olfactory system is unique because the primary neurons
of the olfactory pathway project directly to the cerebral cortex.
As a consequence, the olfactory epithelium allows the influx of
some drugs into the olfactory bulb using axonal transport, and
further movement into the central nervous system. The entry of
drugs into the olfactory bulb is also possible probably by direct
diffusion into the surrounding CSF. The distribution of drugs from
the nasal membrane into the CSF appears to be controlled by a
combination of their molecular properties. For protein or peptides,
the controlling mechanism involves the tight junctions of epithelia
at the outer layer of the olfactory bulbs. The chitosan-shelled
bioactive nanoparticles or fragments of the present invention
possess the molecular properties of enhanced permeating through the
tight junctions as described above. It is generally accepted that
the nasal route circumvents the first-pass liver metabolism and
elimination associated with oral drug delivery. Some aspects of the
invention relate to a method of delivering a bioactive agent into
CSF comprising providing bioactive nanoparticles or fragments
intranasally, wherein the bioactive nanoparticles or fragments
comprise a shell substrate composed mostly of chitosan, a core
substrate that comprises the bioactive agent and a negatively
charged substrate that is at least partially neutralized with a
portion of the positively charged chitosan in the core portion.
[0286] As discussed and disclosed above, positively charged
chitosan has the property to interact with the negatively charged
tight junctions' ZO-1 protein. Therefore, it is suggested that the
drug or bioactive agent loaded chitosan-shelled nanoparticles or
collapsed nanoparticles function as targeting particles toward
tight junctions and release the payload (drug or bioactive agent)
at about the tight junction. This demonstrated that the
nanoparticles with CS dominated on the surfaces could effectively
open or loosen the tight junctions between Caco-2 cells, resulting
in a decrease in the TEER values. It was reported that interaction
of the positively charged amino groups of CS with the negatively
charged sites on tight junctions induces a redistribution of
F-actin and the tight junction's protein ZO-1.
[0287] Some aspects of the present invention relate to a method for
treating disorders or diseases of a tight junction comprising
delivering a pharmaceutical composition of nanoparticles to the
tight junction, wherein the nanoparticles consist of positively
charged chitosan, a negatively charged substrate, optionally a
zero-charge compound, and at least one bioactive agent for treating
the disorders or diseases of the tight junction. In one embodiment,
the nanoparticles are administered via an oral route, a parenteral
route, or a nasal pathway, wherein the nasal pathway may be to
enter an olfactory mucosa, followed by entering a brain tissue via
cerebrospinal fluid (CSF). In another embodiment, the freeze-dried
nanoparticles are being re-constituted with sterile water prior to
being delivered to the tight junction.
[0288] Although the present invention has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims. Many modifications
and variations are possible in light of the above disclosure.
* * * * *