U.S. patent application number 12/008556 was filed with the patent office on 2008-07-03 for nanoparticles for drug delivery.
Invention is credited to Mei-Chin Chen, Yu-Hsin Lin, Hsing-Wen Sung, Hosheng Tu.
Application Number | 20080160078 12/008556 |
Document ID | / |
Family ID | 46325546 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080160078 |
Kind Code |
A1 |
Chen; Mei-Chin ; et
al. |
July 3, 2008 |
Nanoparticles for drug delivery
Abstract
The invention discloses the nanoparticles composed of chitosan,
poly-glutamic acid, and at least one bioactive agent of HMG-CoA
reductase inhibitors or erythropoietin. The nanoparticles are
characterized with a positive surface charge and their enhanced
permeability for paracellular drug delivery.
Inventors: |
Chen; Mei-Chin; (Taipei
County, TW) ; Tu; Hosheng; (Newport Beach, CA)
; Sung; Hsing-Wen; (Hsinchu, TW) ; Lin;
Yu-Hsin; (Kaohsiung, TW) |
Correspondence
Address: |
HOSHENG TU
15 RIEZ
NEWPORT BEACH
CA
92657-0116
US
|
Family ID: |
46325546 |
Appl. No.: |
12/008556 |
Filed: |
January 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11442192 |
May 26, 2006 |
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12008556 |
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11398440 |
Apr 5, 2006 |
7291598 |
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11442192 |
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11284734 |
Nov 21, 2005 |
7282194 |
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11398440 |
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11029082 |
Jan 4, 2005 |
7265090 |
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11284734 |
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Current U.S.
Class: |
424/456 ;
424/493; 514/7.7; 977/906 |
Current CPC
Class: |
A61K 31/722 20130101;
A61K 31/366 20130101; A61K 31/22 20130101; A61K 31/401 20130101;
A61K 47/36 20130101; A61K 31/727 20130101; A61K 47/6939 20170801;
A61K 9/5161 20130101; A61K 9/5146 20130101; B82Y 5/00 20130101;
A61K 47/6931 20170801 |
Class at
Publication: |
424/456 ; 514/8;
424/493; 977/906 |
International
Class: |
A61K 9/48 20060101
A61K009/48; A61K 38/22 20060101 A61K038/22; A61K 9/14 20060101
A61K009/14 |
Claims
1-20. (canceled)
21. A pharmaceutical composition of nanoparticles for oral
administration in a patient, said nanoparticles comprising a shell
portion of biodegradable chitosan that is positively charged, a
core portion of negatively charged substrate that is neutralized
with a portion of positively charged chitosan, and erythropoietin
hormone loaded within said nanoparticles.
22. The pharmaceutical composition of claim 21, wherein said core
portion comprises PGA.
23. The pharmaceutical composition of claim 22, wherein said PGA is
.gamma.-PGA.
24. The pharmaceutical composition of claim 21, wherein said core
portion comprises heparin.
25. The pharmaceutical composition of claim 21, wherein a surface
of said nanoparticles is characterized with a positive surface
charge.
26. The pharmaceutical composition of claim 21, wherein said
nanoparticles have a surface charge from about +15 mV to about +50
mV.
27. The pharmaceutical composition of claim 21, wherein said
nanoparticles are encapsulated in a softgel capsule.
28. The pharmaceutical composition of claim 27, wherein said
softgel capsule is treated with enteric coating.
29. The pharmaceutical composition of claim 21, wherein said
erythropoietin hormone is synthetic erythropoietin.
30. The pharmaceutical composition of claim 21, wherein said
erythropoietin hormone is synthetic erythropoietin produced by
recombinant DNA technology.
31. The pharmaceutical composition of claim 21, wherein said
erythropoietin is characterized as a glycoprotein with a molecular
mass of about 30,000 Daltons.
32. The pharmaceutical composition of claim 21, wherein said
erythropoietin hormone is a long-acting darbepoetin.
33. The pharmaceutical composition of claim 21, wherein said
erythropoietin hormone is erythropoiesis-stimulating protein.
34. The pharmaceutical composition of claim 21, wherein said
nanoparticles are formed via a simple and mild ionic-gelation
method.
35. A method of delivering erythropoietin hormone to blood
circulation in a patient, comprising: providing nanoparticles
according to the pharmaceutical composition of claim 21;
administering said nanoparticles orally toward an intestine of the
patient; urging said nanoparticles to pass through an epithelial
barrier of the intestine; and releasing said erythropoietin hormone
into the blood circulation.
36. The method of claim 35, wherein said core portion of the
nanoparticles comprises PGA.
37. The method of claim 36, wherein said PGA is .gamma.-PGA.
38. The method of claim 35, wherein said core portion of the
nanoparticles comprises heparin.
39. The method of claim 35, wherein said nanoparticles are
encapsulated in a softgel capsule.
40. The method of claim 39, wherein said softgel capsule is treated
with enteric coating.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/442,192, filed May 26, 2006, which
is a continuation-in-part application of U.S. patent application
Ser. No. 11/398,440, filed Apr. 5, 2006, now U.S. Pat. No.
7,291,598 B2, issued Nov. 6, 2007, 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 B2, issued Oct. 16,
2007, 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 B2, issued Sep. 4, 2007, the entire contents of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to formulation and medical
uses of biodegradable nanoparticles with therapeutic bioactive
agents and their oral delivery having enhanced paracellular
permeability.
BACKGROUND OF THE INVENTION
[0003] Production of pharmaceutically active peptides and proteins
in large quantities has become feasible (Biomacromolecules 2004;
5:1917-1925). The oral route is considered the most convenient way
of drug administrations for patients. 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). 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 as with the
epithelial cells of the gastrointestinal tract, is termed
paracellular transport. Paracellular transport is passive.
Paracellular transport depends on electrochemical gradients
generated by transcellular transport and on 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 "tightness" 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 good 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 that is
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 could not partition into the hydrophobic
membrane and thus can only traverse 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 feature 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 that is sometimes 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] 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 was reported from
literature that this naturally occurring .gamma.-PGA is a
water-soluble, biodegradable, and non-toxic polymer. A related, but
structurally different polymer, [poly(.alpha.-glutamic acid),
.alpha.-PGA] has been used for drug delivery (Adv. Drug Deliver.
Rev. 2002; 54:695-713; Cancer Res. 1998; 58:2404-2409). A-PGA is
usually synthesized from poly(.gamma.-benzyl-L-glutamate) by
removing the benzyl protecting group with the use of hydrogen
bromide. Hashida et al. used .alpha.-PGA as a polymeric backbone
and galactose moiety as a ligand to target hepatocytes (J. Control.
Release 1999; 62:253-262). Their in vivo results indicated that the
galactosylated .alpha.-PGA had a remarkable targeting ability to
hepatocytes and degradation of .alpha.-PGA was observed in the
liver.
[0009] 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.
[0010] U.S. Patent Application publication no. 2006/0051424A1,
published on Mar. 9, 2006, entire contents of which are
incorporated herein by reference, discloses nanoparticle
compositions comprising a cationic biopolymer and at least one
biologically active substance, pharmaceutical compositions
comprising such nanoparticles and methods for the oral
administration of biologically active molecules which are
susceptible to degradation in the gastro-intestinal tract using
nanoparticle.
[0011] U.S. Patent Application publication no. 2005/0042298A1,
published on Feb. 24, 2005, entire contents of which are
incorporated herein by reference, discloses a functionalized
composition for use in forming an immunonanoparticle, the
functionalized composition comprising a nanoparticle-forming
polymer, a polymeric strand having a first end attached to the
nanoparticle-forming polymer and a second end, and a conjugation
agent attached to the second end of the polymeric strand. In one
embodiment, a functionalized moiety for use in forming the
immunonanoparticle further includes a targeting agent attached to
the conjugation agent.
[0012] U.S. Pat. No. 6,689,338 B2 issued on Feb. 10, 2004, entire
contents of which are incorporated herein by reference, discloses a
bioconjugate comprising a radioactive, metal sulfide or metal oxide
nanoparticle covalently linked to at least one biological vector
molecule, wherein the at least one biological vector molecule is
selected from a group consisting of monoclonal antibodies (mAb),
fragments of monoclonal antibodies, and peptides.
[0013] U.S. Pat. No. 6,165,440, issued on Dec. 26, 2000, entire
contents of which are incorporated herein by reference, discloses a
method of anti-cancer drug delivery in a solid tumor, comprising
the steps of administering at least one anti-cancer drug to the
tumor, injecting nanoparticles or microparticles to the tumor
intravenously, and irradiating the tumor with radiation, wherein
the anti-cancer drug is selected from the group consisting of a
monoclonal antibody, a cytokine, an antisense oligonucleotide, and
a gene-targeting vector.
[0014] U.S. Pat. No. 6,777,552, issued on Aug. 17, 2004, entire
contents of which are incorporated herein by reference, discloses
processes for preparing a calcium salt of a statin from an ester
derivative or protected ester derivative of the statin by using
calcium hydroxide.
[0015] Currently, most statin is administered to a patient orally
on a daily basis. It is desirable to administer statin or other
therapeutic drugs orally in a nanoparticle form that provide
enhanced paracellular permeability, bioavailability, and sustained
release over an extended period, where the nanoparticles biodegrade
to biocompatible byproducts in situ.
SUMMARY OF THE INVENTION
[0016] It is one object of the present invention to provide a novel
nanoparticle system and methods of preparation for paracellular
transport drug delivery using a simple and mild ionic-gelation
method upon addition of a poly-.gamma.-glutamic acid (.gamma.-PGA)
solution into a low molecular weight chitosan (low-MW CS) solution.
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
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.
[0017] The results obtained by the TEM (transmission electron
microscopy) and AFM (atomic force microscopy) examinations showed
that the morphology of the prepared nanoparticles was generally
spherical in shape. Evaluation of the prepared nanoparticles in
enhancing intestinal paracellular transport was investigated in
vitro in Caco-2 cell monolayers. In some aspects of the present
invention, it provides the nanoparticles with CS dominated on the
surfaces to effectively reduce the transepithelial electrical
resistance (TEER) of Caco-2 cell monolayers. The confocal laser
scanning microscopy (CLSM) observations confirm that the
nanoparticles with CS dominated on the surface are able to open the
tight junctions between Caco-2 cells and allows transport of the
nanoparticles via the paracellular pathways.
[0018] In some application, a normal or high molecular weight
chitosan is used in preparing the nanoparticles.
[0019] Some aspects of the invention relate to a method of
enhancing intestinal or blood brain paracellular transport
configured for delivering at least one bioactive agent in a patient
comprising administering nanoparticles composed of .gamma.-PGA and
chitosan, wherein the step of administering the nanoparticles may
be via oral administration. In one embodiment, the chitosan
dominates on a surface of the nanoparticles as shell substrate and
the negatively charged .gamma.-PGA as core substrate. In another
embodiment, a substantial surface of the nanoparticles is
characterized with a positive surface 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, at least one
bioactive or protein drug is conjugated with the negatively charged
core substrate.
[0020] In a further embodiment, the chitosan of the nanoparticles
is a low molecular weight chitosan, wherein the low molecular
weight chitosan has a molecular weight of about 50 kDa, preferably
having a molecular weight of less than about 40 kDa.
[0021] In a further embodiment, the nanoparticles have a mean
particle size between about 50 and 400 nanometers, preferably
between about 100 and 300 nanometers, and most preferably between
about 100 and 200 nanometers.
[0022] 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 a group consisting of
synthetic drugs, proteins (for example, monoclonal antibodies),
peptides, nucleosides, nucleotides, antiviral agents,
antineoplastic agents, antibiotics, and anti-inflammatory drugs.
Further, the bioactive agent may be selected from a 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-II, interferon, colony
stimulating factor, tumor necrosis factor (TNF) and
melanocyte-stimulating hormone. In one preferred embodiment, the
bioactive agent is an Alzheimer antagonist.
[0023] Some aspects of the invention relate to an oral dose of
nanoparticles that effectively enhance intestinal or blood brain
paracellular transport comprising .gamma.-PGA or .alpha.-PGA 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
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,
wherein the chitosan dominates on a surface of the nanoparticles
with positive charges. 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), and 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).
[0024] Some aspects of the invention relate to an oral dose of
nanoparticles that effectively enhance intestinal or blood brain
paracellular transport comprising .gamma.-PGA and low molecular
weight chitosan, wherein the nanoparticles are crosslinked with a
crosslinking agent or with light, such as ultraviolet
irradiation.
[0025] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, each nanoparticle comprising 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 third component
is .gamma.-PGA, .alpha.-PGA, derivatives or salts of PGA, heparin,
glycosaminoglycans 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.
[0026] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, each nanoparticle comprising 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, wherein 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. 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, and anti-inflammatory drugs.
[0027] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, wherein the nanoparticles are further
encapsulated in a gelcap.
[0028] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, each nanoparticle comprising 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, wherein the second component is
crosslinked. In one embodiment, the degree of crosslinking is less
than 50%. In another embodiment, the degree of crosslinking is
ranged between 1% and 20%.
[0029] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, each nanoparticle comprising 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, wherein the second component is
crosslinked with a crosslinking agent selected from the group
consisting of genipin, its derivatives, analog, stereoisomers and
mixtures thereof. In one embodiment, the crosslinking agent is
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.
[0030] Some aspects of the invention provide a dose of
nanoparticles characterized by enhancing intestinal or brain blood
paracellular transport, wherein the low molecule weight chitosan
has a molecular weight of 80 kDa or less. In one embodiment, the
low molecule weight chitosan is further grafted with a polymer
having a chemical formula as:
##STR00001##
where R is .gtoreq.12
[0031] Some aspects of the invention provide a method of enhancing
intestinal or brain blood paracellular transport 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 intestinal paracellular transport. In
another embodiment, the step of administering the dose of
nanoparticles is via venous administration or injection for
enhancing brain blood paracellular transport or reducing the
blood-brain barrier (BBB).
[0032] Some aspects of the invention provide a method of treating
diabetes of a patient 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 shell
substrate comprises chitosan, chitin, chitosan oligosaccharides,
and chitosan derivatives thereof, wherein a substantial portion of
a surface of the nanoparticles is characterized with a positive
surface charge. In another embodiment, the core substrate is
selected from a group consisting of .gamma.-PGA, .alpha.-PGA,
water-soluble salts of PGA, metal salts of PGA, heparin, heparin
analogs, low molecular weight heparin, glycosaminoglycans, and
alginate. The molecular formula of the insulin is selected from a
group consisting of C.sub.254H.sub.377N.sub.65O.sub.75S.sub.6,
C.sub.257H.sub.383N.sub.65O.sub.77S.sub.6,
C.sub.256H.sub.381N.sub.65O.sub.79S.sub.6,
C.sub.267H.sub.404N.sub.72O.sub.78S.sub.6, and the like.
[0033] In one embodiment, the orally administering insulin
containing nanoparticles comprise a dosage effective amount of the
insulin to treat the diabetes comprising an insulin amount of
between about 15 units to 45 units, preferably between about 25
units to 35 units, per kilogram body weight of the patient. In a
further embodiment, the insulin-containing nanoparticle comprises a
trace amount of zinc or calcium, or is treated with enteric
coating.
[0034] In one embodiment, the insulin containing nanoparticles
further comprise at least one paracellular transport enhancer,
wherein the paracellular transport enhancer may be selected from a
group consisting of Ca.sup.2+ chelators, bile salts, anionic
surfactants, medium-chain fatty acids, and phosphate esters. In
another embodiment, the nanoparticles and the paracellular
transport enhancer are co-encapsulated in a softgel capsule or are
encapsulated separately.
[0035] Some aspects of the invention provide nanoparticles for oral
administration in a patient, comprising a positively charged shell
substrate, a negatively charged core substrate, and a bioactive
agent conjugated with the core substrate, wherein the core
substrate is selected from a group consisting of heparin, heparin
analogs, low molecular weight heparin, glycosaminoglycans, and
alginate, the bioactive agent being selected from a group
consisting of chondroitin sulfate, hyaluronic acid, growth factor
and protein with pharmaceutically effective amount.
[0036] Some aspects of the invention provide nanoparticles for oral
administration in a patient, comprising a positively charged shell
substrate, a negatively charged core substrate, and a bioactive
agent conjugated with the core substrate, wherein the bioactive
agent is calcitonin or vancomycin.
[0037] Some aspects of the invention provide a method of treating
Alzheimer's diseases of a patient comprising intravenously
administering bioactive nanoparticles with a dosage effective to
treat the Alzheimer's diseases, wherein the bioactive nanoparticles
comprises a positively charged shell substrate, a negatively
charged core substrate, and at least one bioactive agent for
treating Alzheimer's disease, wherein the at least one bioactive
agent is selected from a group consisting of memantine
hydrochloride, donepezil hydrochloride, rivastigmine tartrate,
galantamine hydrochloride, and tacrine hydrochloride.
[0038] In one embodiment, the dosage effective to treat the
Alzheimer's diseases comprises administering the at least one
bioactive agent for treating Alzheimer's disease at about 10 mg to
40 mg per day over a period of one month to one year. In another
embodiment, at least a portion the shell substrate is crosslinked,
preferably at a degree of crosslinking less than about 50%, or most
preferably between about 1% and 20%.
[0039] Some aspects of the invention provide a method of treating a
target tissue or organ of a patient with a monoclonal antibody,
comprising the steps of: providing the monoclonal antibody to the
tissue or organ, wherein the monoclonal antibody is encapsulated
within nanoparticles; administering the nanoparticles to the
patient orally; and treating the target tissue or organ with the
monoclonal antibody that is sustained released from the
nanoparticles. In one embodiment, the monoclonal antibody is an
anti-cancer drug. In another embodiment, the monoclonal antibody is
Adalimumab or Bevacizumab.
[0040] Some aspects of the invention provide nanoparticles for oral
administration in a patient, comprising a positively charged shell
substrate, a negatively charged core substrate, and a monoclonal
antibody encapsulated within the shell substrate. In one preferred
embodiment, the monoclonal antibody is mixed with, conjugated or
coupled to, the core substrate.
[0041] Some aspects of the invention provide a method of delivering
an HMG-CoA reductase inhibitor to blood circulation in a patient,
comprising: (a) providing nanoparticles that encapsulate the
HMG-CoA reductase inhibitor, wherein the nanoparticles are
biodegradable; (b) administering the nanoparticles orally that move
toward an intestine of the patient; (c) urging the nanoparticles to
pass through an epithelial barrier of the intestine; and (d)
releasing the HMG-CoA reductase inhibitor into the blood
circulation through the capillaries surrounding the exterior
surface of the intestine in a sustained manner.
[0042] Some aspects of the invention provide a pharmaceutical
composition of nanoparticles for oral administration in a patient,
comprising a biodegradable chitosan shell substrate, and a HMG-CoA
reductase inhibitor encapsulated within the shell substrate,
wherein the released HMG-CoA reductase inhibitor is
pharmaceutically effective. In one embodiment, the HMG-CoA
reductase inhibitor is released into blood circulation in a
sustained manner. In another embodiment, the HMG-CoA reductase
inhibitor is released via diffusion, biodegradation of the shell
substrate, or both.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] 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.
[0044] 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.
[0045] FIG. 2 shows (a) FT-IR and (b) .sup.1H-NMR spectra of the
purified .gamma.-PGA obtained from microbial fermentation.
[0046] FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared
CS-.gamma.-PGA nanoparticles.
[0047] 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).
[0048] 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.
[0049] FIG. 6 shows effects of the prepared CS-.gamma.-PGA
nanoparticles on the TEER values of Caco-2 cell monolayers.
[0050] 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.
[0051] FIG. 8 shows an illustrative protein transport mechanism
through a cell layer, including transcellular transport and
paracelluler transport.
[0052] FIG. 9 shows a schematic illustration of a paracellular
transport mechanism.
[0053] FIG. 10 shows an fCS-.gamma.-PGA nanoparticle with
FITC-labeled chitosans having positive surface charge.
[0054] FIG. 11 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan and .gamma.-PGA.
[0055] FIG. 12 shows loading capacity and association efficiency of
insulin in nanoparticles of chitosan as reference.
[0056] FIG. 13 shows the stability of insulin-loaded
nanoparticles.
[0057] FIG. 14 shows a representative in vitro study with insulin
drug release profile in a pH-adjusted solution.
[0058] FIG. 15 shows the bioavailability of insulin of orally
administered insulin-loaded nanoparticles in diabetic rats.
[0059] FIG. 16 shows a proposed mechanism of nanoparticles released
from the enteric coating.
[0060] FIG. 17 shows the schematic illustration of insulin
conjugated with histidine and/or glutamic acid side groups of the
.gamma.-PGA via zinc.
[0061] FIG. 18 shows the schematic illustration of insulin
conjugated with a carboxyl side group of the .gamma.-PGA via
zinc.
[0062] FIG. 19 shows a schematic composition of a nanoparticle with
a shell substrate and a core substrate having a statin (HMG-CoA
reductase inhibitor).
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0063] The preferred embodiments of the present invention described
below relate particularly to 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 will
be appreciated 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.
[0064] .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
[0065] 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 (HBSS) 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
[0066] 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 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).
[0067] 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 0.01 M
NaH.sub.2PO.sub.4 and 0.5 M NaNO.sub.3 and was brought to a pH of
2.0. The flow rate of mobile phase was 1.0 ml/min, and the columns
and the R1 detector cell were maintained at 30.degree. C.
[0068] Factors limiting applications of most commercially available
CSs are their high molecular weight and thus 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).
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. Thus obtained 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.
[0069] 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).
Example No. 3
Production and Purification of .gamma.-PGA
[0070] .gamma.-PGA was produced by Bacillus licheniformis (ATCC
9945, Bioresources Collection and Research Center, Hsinchu, Taiwan)
as per a method reported by Yoon et al. with slight modifications
(Biotechnol. Lett. 2000; 22:585-588). Highly mucoid colonies (ATCC
9945a) were selected from Bacillus licheniformis (ATCC 9945)
cultured on the E medium (ingredients comprising L-glutamic acid,
20.0 g/l; citric acid, 12.0 g/l; glycerol, 80.0 g/l; NH.sub.4Cl,
7.0 g/l; K.sub.2HPO.sub.4, 0.5 g/l; MgSO.sub.4.7H.sub.2O, 0.5 g/l;
FeCl.sub.3.6H.sub.2O, 0.04 g/l; CaCl.sub.2.2H.sub.2O, 0.15 g/l;
MnSO.sub.4.H.sub.2O, 0.104 g/l, pH 6.5) agar plates at 37.degree.
C. for several times. Subsequently, young mucoid colonies were
transferred into 10 ml E medium and grown at 37.degree. C. in a
shaking incubator at 250 rpm for 24 hours. Afterward, 500 .mu.l of
culture broth was mixed with 50 ml E medium and was transferred
into a 2.5-1 jar-fermentor (KMJ-2B, Mituwa Co., Osaka, Japan)
containing 950 ml of E medium. Cells were cultured at 37.degree. C.
The pH was controlled at 6.5 by automatic feeding of 25% (v/v)
NH.sub.4OH and/or 2M HCl. The dissolved oxygen concentration was
initially controlled at 40% of air saturation by supplying air and
by controlling the agitation speed up to 1000 rpm.
[0071] After 40 hours, cells were separated from the culture broth
by centrifugation for 20 minutes at 12,000.times.g at 4.degree. C.
The supernatant containing .gamma.-PGA was poured into 4 volumes of
methanol and left overnight with gentle stirring. The resulting
precipitate containing crude .gamma.-PGA was collected by
centrifugation for 40 minutes at 12,000.times.g at 4.degree. C. and
then was dissolved in deionized water to remove insoluble
impurities by centrifugation for 20 minutes at 24,000.times.g at
4.degree. C. The aqueous .gamma.-PGA solution was desalted by
dialysis (MWCO: 100,000, Spectrum Laboratories, Inc., Laguna Hills,
Calif.) against distilled water for 12 hours with water exchanges
several times, and finally was lyophilized to obtain pure
.gamma.-PGA.
[0072] The purified .gamma.-PGA was verified by the proton nuclear
magnetic resonance (.sup.1H-NMR) and the FT-IR analyses. Analysis
of .sup.1H-NMR was conducted on an NMR spectrometer (Varian
Unityionva 500 NMR Spectrometer, MO) using DMSO-d.sub.6 at 2.49 ppm
as an internal reference. Test samples used for the FT-IR analysis
first were dried and ground into a powder form. The powder then was
mixed with KBr (1:100) and pressed into a disk. Analysis was
performed on an FT-IR spectrometer (Perkin Elmer Spectrum RX1 FT-IR
System, Buckinghamshire, England). The samples were scanned from
400-4000 cm.sup.-1. The average molecular weight of the purified
.gamma.-PGA was determined by the same GPC system as described
before. Polyethylene glycol (molecular weights of 106-22,000) and
polyethylene oxide (molecular weights of 20,000-1,000,000, PL
Laboratories) standards were used to construct a calibration curve.
The mobile phase contained 0.01M NaH.sub.2PO.sub.4 and 0.2M
NaNO.sub.3 and was brought to a pH of 7.0.
[0073] The purified .gamma.-PGA obtained from fermentation 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. 4
Preparation of the Cs-.gamma.-PGA Nanoparticles
[0074] 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. 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.
[0075] As stated, nanoparticles were obtained instantaneously upon
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).
Example No. 5
Characterization of the CS-.gamma.-PGA Nanoparticles
[0076] The morphological examination of the CS-.gamma.-PGA
nanoparticles was performed by TEM (transmission electron
microscopy) and AFM (atomic force microscopy). The TEM sample was
prepared by placing a drop of the nanoparticle solution onto a 400
mesh copper grid coated with carbon. About 2 minutes after
deposition, the grid was tapped with a filter paper to remove
surface water and positively stained by using an alkaline bismuth
solution (Microbiol. Immunol. 1986; 30:1207-1211). The AFM sample
was prepared by casting a drop of the nanoparticle solution on a
slide glass and then dried in vacuum. The size distribution and
zeta potential of the prepared nanoparticles were measured using a
Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire,
UK).
[0077] During storage, aggregation of nanoparticles may occur and
thus leads to losing their structural integrity or forming
precipitation of nanoparticles (Eur. J. Pharm. Sci. 1999;
8:99-107). Therefore, the stability of nanoparticles during storage
must be evaluated. In the stability study, the prepared
nanoparticles suspended in deionized water (1 mg/ml) were stored at
4.degree. C. and their particle sizes and zeta potential values
were monitored by the same Zetasizer as mentioned earlier during
storage.
[0078] In the preparation of nanoparticles, samples were visually
analyzed and three distinct solution systems were identified: clear
solution, opalescent suspension, and solution with precipitation of
aggregates. Examined by the Zetasizer, nanoparticles were found in
the clear solution and the opalescent suspension rather than in the
solution with precipitation of aggregates.
[0079] The particle sizes and the zeta potential values of
CS-.gamma.-PGA nanoparticles, prepared at varying concentrations of
Y-PGA and CS, were determined and the results are shown in Tables
1a and 1b. 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
.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 interacting with more CS molecules, and thus formed a
lager size of nanoparticles (Table 1a, p<0.05). When the amount
of CS molecules exceeded that of local .gamma.-PGA molecules, some
of the excessive CS molecules were entangled onto the surfaces of
CS-.gamma.-PGA nanoparticles.
[0080] Thus, the resulting nanoparticles may display a structure of
a neutral polyelectrolyte-complex core surrounded by a positively
charged CS shell (Table 1b) ensuring the 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,
preferably between about 100 and 300 nanometers, and most
preferably between about 100 and 200 nanometers. The morphology of
the nanoparticles shows 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.
[0081] 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 for up to 6 weeks. It was
found that neither aggregation nor precipitation of nanoparticles
was observed during storage for up to 6 weeks, as a result of the
electrostatic repulsion between the positively charged
CS-.gamma.-PGA nanoparticles (for the former group) or the
negatively charged CS-.gamma.-PGA nanoparticles (for the latter
group).
[0082] 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.
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
Example No. 6
Caco-2 Cell Cultures and TEER Measurements
[0083] 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).
[0084] TEER values of the Caco-2 cell monolayers were monitored
with a Millicell.RTM.-Electrical Resistance System (Millipore
Corp., Bedford, Mass.) connected to a pair of chopstick electrodes.
To initiate the transport experiments, the culture media in the
donor and acceptor compartments were aspirated, and the cells were
rinsed twice with pre-warmed transport media (HBSS supplemented
with 25 mM glucose, pH 6.0). Following a 30-min equilibration with
the transport media at 37.degree. C., the cells were incubated for
2 hours with 2 ml transport media containing 0.5 ml test
nanoparticle solutions (0.2 mg/ml) at 37.degree. C. Subsequently,
solutions of nanoparticles were carefully removed and cells were
washed three times with HBSS and replaced by fresh culture media.
The TEER was measured for another 20 hours to study reversibility
of the effect of test nanoparticles on Caco-2 cell monolayers (Eur.
J. Pharm. Sci. 2000; 10:205-214).
[0085] 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
was 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.
[0086] 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 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.
[0087] 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.
[0088] 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 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. The mucoadhesive CS-shelled
nanoparticles can prolong their residence in the intestine and
mediate transient opening of the tight junctions of epithelial
cells, thereby facilitating the encapsulated nanoparticles crossing
the epithelium barrier and enhancing the intestinal absorption of
drugs.
Example No. 7
fCS-.gamma.-PGA Nanoparticle Preparation and CLSM Visualization
[0089] Fluorescence (FITC)-labeled CS-.gamma.-PGA (fCS-.gamma.-PGA)
nanoparticles (FIG. 10) 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 was then dialyzed for 3
days in the dark against 5 liters of distilled water, with water
replaced on a daily basis. The resultant fCS was lyophilized in a
freeze dryer. The fCS-.gamma.-PGA nanoparticles were prepared as
per the procedure described in Example No. 4.
[0090] Subsequently, 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 for 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).
[0091] 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).
[0092] After 60 minutes of incubation with the nanoparticles, the
intensity of fluorescence observed at intercellular spaces was
stronger and appeared at a deeper level than those observed at 20
min after incubation. These observations confirmed with our TEER
results 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 allowed transport of the nanoparticles by
passive diffusion via the paracellular pathways.
Example No. 8
In Vivo Study with Fluorescence-Labeled Nanoparticles
[0093] 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 are sacrificed at a pre-determined time and
the intestine is isolated for CLSM examination. The fluorescence
images of the nanoparticles were clearly observed by CLSM that
penetrates 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.
Example No. 9
Insulin Loading Capacity in Nanoparticles
[0094] Fluorescence (FITC)-labeled .gamma.-PGA was added into
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 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.
[0095] 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 >27 USP units/mg. The insulin
contains 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 and 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 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.
[0096] 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 in 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.
[0097] 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 made 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) than regular insulin or the
intermediate-acting insulins (NPH and Lente).
[0098] 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 and
a molecular weight of 6063.
[0099] 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.
[0100] 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.
[0101] 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 2
below.
TABLE-US-00003 TABLE 2 Insulin Conc. (mg/ml) Mean Particle
Polydispersity Zeta Potential (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
[0102] 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 ( AE % ) Loading Capacity ( LC ) = ( Total amount
insulin - Insulin in superatant ) Weight of recovered particles
.times. 100 % ##EQU00001.2##
[0103] 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 AE (40.about.55%) and LC (5.0.about.14.0%) of insulin for
CS-.gamma. PGA nanoparticles was obtained by using ionic-gelation
method upon addition of insulin mixed with .gamma.-PGA solution
into CS solution, followed by magnetic stirring for nanoparticle
separation. Some aspects of the invention relate to an oral dose of
nanoparticles that effectively enhance 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.
Example No. 10
Insulin Loading in PGA Nanoparticles
[0104] The nanoparticles with two core substrates (.gamma.-PGA and
.alpha.-PGA) are prepared at a chitosan to PGA ratio of 0.75 mg/ml
to 0.167 mg/ml with insulin concentration at 0.083 mg/ml (sample
size, n=3). Their particle size, zeta potential, and insulin
loading efficiency are quite comparable and are shown in Table 3
below.
TABLE-US-00004 TABLE 3 Zeta Loading Core Mean Particle
Polydispersity Potential Efficiency Substrate Size (nm) Index (PI)
(mV) (%) .gamma.-PGA 218.4 .+-. 5.2 0.32 .+-. 0.09 +25.4 .+-. 1.22
52.1 .+-. 4.3 .alpha.-PGA 207.6 .+-. 9.3 0.29 .+-. 0.07 +26.8 .+-.
1.01 59.1 .+-. 5.0
[0105] Some aspects of the invention relate to a dose of
nanoparticles that effectively enhance intestinal 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 a surface of the
nanoparticles with surface positive charges. In use, firstly,
encapsulate the Alzheimer's drug in the chitosan shell nanoparticle
as described herein, wherein the nanoparticle is partially
crosslinked (optionally) to enhance its biodurability. Then
intra-venously inject the nanoparticles, 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 after passing
the tight junction for therapeutic treatment. 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.
[0106] 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 a patient 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 a 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).
[0107] 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 form of polyglutamic acid may be selected from a
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##
[0108] Some aspects of the invention relate to the negatively
charged glycosaminoglycans (GAGs) as the core substrate of the
present nanoparticles. GAGs may be used to complex with a
low-molecular-weight chitosan to form drug-carrier nanoparticles.
GAGs may also conjugate with the proteins (for example, monoclonal
antibodies) 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.
[0109] 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 sustained released from these tablets within 5 hours. 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 observation of 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.
[0110] Pan et al. disclosed 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 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. 11
Insulin Nanoparticle Stability
[0111] 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 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
containing a dosage of insulin nanoparticles effective amount of
the insulin to treat or manage the diabetic patients, 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. 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 the single dosage forms.
[0112] 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, and suspended in a liquid solution
and the like. The nanoparticles can be suspended in a deionized
solution or the like 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 are 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 soft gel 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.
Example No. 12
In Vitro Insulin Release Study
[0113] 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.
[0114] 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. To further simulate the
exit portion of the intestine, the insulin-containing nanoparticle
solution is adjusted to pH 7.4. The remaining insulin (about 67%)
is released from the nanoparticles. 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 nanoparticles of the present invention with chitosan at the
outer surface (preferential mucosal adhesion on the intestinal
wall) and positive charge (enhancing paracellular tight junction
transport).
Example No. 13
In Vivo Study with Insulin-Loaded Fluorescence-Labeled
Nanoparticles
[0115] 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 rats. 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) is
measured at 107.2.+-.8.1 mg/dL for normal rats while the blood
glucose level is 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; (b) 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.
[0116] 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 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.
[0117] 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 at 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 a patient
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
patient, preferably 20 to 40 units, and most preferably at about 25
to 35 units insulin per kilogram body weight.
[0118] 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 human
patient.
[0119] Exemplary drugs of the present invention 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; 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.
[0120] The bioactive agent of the present invention may 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.
[0121] 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 at an outer layer and
surface positive charge serve as an enhancer in enhancing
paracellular drug (bioactive agent) transport 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. 14
Paracellular Transport and Enhancers
[0122] Chitosan and its derivatives may function as intestinal
absorption enhancers (that is, paracellular transport enhancers).
Chitosan, when protonated at an acidic pH, is able to increase the
paracellular 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
paracellular transport enhancer (in non-nanoparticle form or
nanoparticle form). In one embodiment, the nanoparticles can be
formulated by co-encapsulation of the at least one paracellular
transport enhancer and at least one bioactive agent, optionally
with other components. The 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.
[0123] In some embodiment, the nanoparticles of the present
invention and the at least one paracellular transport enhancer are
encapsulated in a soft gel, pill, or enteric coated. The enhancers
and the nanoparticles would arrive at the tight junction about the
same time for enhancing opening the tight junction. In another
embodiment, the at least one paracellular transport enhancer is
co-enclosed within the shell of the nanoparticles of the present
invention. Therefore, some broken nanoparticles 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. 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 intestinal tract to assist the
drug-containing first nanoparticles to open and pass the tight
junction.
Example No. 15
Nanoparticles with Complexed Calcitonin
[0124] Calcitonin is a protein drug that serves therapeutically 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 is suitable to complex or
conjugate with negatively charged core substrate, such as
.gamma.-PGA or .alpha.-PGA. In preparation, nanoparticles were
obtained upon addition of a mixture of .gamma.-PGA plus calcitonin
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 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 totally
or substantially totally encapsulated in the nanoparticles.
Supernatants were discarded and nanoparticles were resuspended in
deionized water as the solution products, further encapsulated in
soft gels or further treated with an enteric coating.
[0125] Preotact.RTM. (parathyroid hormone) has been demonstrated to
increase bone size and mineral content, thereby improving bone
quality and strength. These biological actions are mediated through
binding to at least two distinct high-affinity cell-surface
receptors specific for the N-terminal and C-terminal regions of the
molecule, both of which are required for normal bone metabolism.
The N-terminal portion of the molecule is primarily responsible for
the bone building effects of parathyroid hormone. The C-terminal
portion of the molecule has antiresorptive activity and is
necessary for normal regulation of N-terminal fragment activity.
The normal physiological role of parathyroid hormone (rDNA origin)
is to regulate calcium and phosphate homeostasis. Actions of
parathyroid hormone include regulation of bone metabolism, renal
tubular reabsorption by stimulating the renal production of the
active metabolite of vitamin D. Preotact.RTM. is injected with the
specially designed and patient focused Preotact.TM. Pen.
[0126] Parathyroid hormone (PTH) is secreted by the parathyroid
glands as a polypeptide containing 84 amino acids. It acts to
increase the concentration of calcium in the blood, whereas
calcitonin (a hormone produced by the thyroid gland) acts to
decrease calcium concentration. PTH acts to increase the
concentration of calcium in the blood in three ways. It enhances
the release of calcium from the large reservoir contained in the
bones, enhances reabsorption of calcium from renal tubules; and
enhances the absorption of calcium in the intestine by increasing
the production of vitamin D and upregulating the enzyme responsible
for 1-alpha hydroxylation of 25-OH vitamin D converting vitamin D
to its active form (1,25-OH vitamin D) which effects the actual
absorption of calcium by the intestine.
[0127] PTH also acts to decrease the concentration of phosphate in
the blood, primarily by reducing reabsorption in the proximal
tubules of the kidney. Increased calcium concentration in the blood
acts (via feedback inhibition) to decrease PTH secretion by the
parathyroid glands. This is achieved by the activation of
calcium-sensing receptors located on parathyroid cells.
Preotact.RTM. (or Preos.RTM., a U.S. version) is recombinant
full-length human parathyroid hormone (PTH 1-84).
[0128] Some aspects of the invention relate to a pharmaceutical
composition of nanoparticles for oral administration in a patient,
comprising a biodegradable chitosan shell substrate, and a
parathyroid hormone encapsulated within the shell substrate. In one
embodiment, the parathyroid hormone is polypeptide containing 84
amino acids that is recombinant full-length human parathyroid
hormone (PTH 1-84).
[0129] Erythropoietin
[0130] Erythropoietin (EPO) is a hormone that is a cytokine for
erythrocyte (red blood cell) precursors in the bone marrow. It is
produced by the kidney, and is the hormone regulating red blood
cell production. After being released into the blood stream it
binds with receptors (EpoR) on the surface or red cell precursors
in the bone marrow, where it stimulates the production of red blood
cells. Synthetic erythropoietin is available as a therapeutic agent
produced by recombinant DNA technology (rEPO). It is used in
treating anemia resulting from chronic renal failure or from cancer
chemotherapy. EPO has now been identified as a glycoprotein with a
molecular mass of about 30,000 Daltons. It has a 165 amino acid
chain with four oligosaccharide side chains and circulates in the
blood plasma at a very low concentration (about 5 pmol/L).
Erythropoietin, an acidic glycoprotein of approximately 34,000
molecular weight, may occur in three forms: .alpha., .beta., and
asialo. The .alpha. and .beta. forms differ slightly in
carbohydrate components, but have the same potency, biological
activity and molecular weight. The asialo form is an .alpha. or
.beta. form with the terminal carbohydrate (sialic acid)
removed.
[0131] A longer-acting erythropoietin analogue, darbepoetin (dEPO),
also known as novel erythropoiesis-stimulating protein (NESP), has
a slightly different amino acid sequence and a greater number of
oligosaccharide residues, relative to rEPO. EPO is generally
injected subcutaneously (under the skin) by the patient, although
it may also be given intravenously. Some aspects of the invention
relate to a pharmaceutical composition of nanoparticles for oral
administration in a patient, comprising a biodegradable chitosan
shell substrate, and a rEPO or dEPO encapsulated within the shell
substrate, wherein the rEPO or dEPO is sustained released into
blood circulation via intestinal paracellular permeation.
Example No. 16
Nanoparticles with Encapsulated Erythropoietin
[0132] In product formulation, nanoparticles were obtained upon
addition of erythropoietin (in one example for illustration, rEPO)
aqueous solution, using a pipette into a low-MW CS aqueous solution
with excess CS concentrations under magnetic stirring at room
temperature. Nanoparticles were collected by ultracentrifugation.
Nanoparticles comprise positively charged shell substrate chitosan
and negatively charged core substrate erythropoietin. The
erythropoietin is substantially or totally encapsulated in the
nanoparticles. In other words, the erythropoietin component is
substantially maintained within the intact nanoparticles during the
nanoparticle delivery phase orally. Thus, erythropoietin does not
cause any significant effect until the nanoparticles dissociate or
biodegrade to release the core contents in a sustained release
manner into the blood stream.
Example No. 17
Nanoparticles Loaded with Vancomycin
[0133] Vancomycin is a protein drug that serves therapeutically as
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 that 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 plus 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 in the nanoparticles.
Supernatants were discarded and nanoparticles were resuspended in
deionized water as the solution products, further encapsulated in
soft gels or further treated with an enteric coating.
[0134] Some aspects of the invention relate to a method of
enhancing intestinal or blood brain paracellular transport of
bioactive agents configured and adapted for delivering at least one
bioactive agent in a patient 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, 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-II (IL2), interferon,
colony stimulating factor (CSF), tumor necrosis factor (TNF) and
melanocyte-stimulating hormone. In a further embodiment, the
bioactive agent is an Alzheimer antagonist.
Example No. 18
Nanoparticles with Heparin Core Substrate
[0135] Heparin is a negatively charged drug that serves
therapeutically as 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 conjugate at least one bioactive
agent as disclosed herein. In preparation, nanoparticles were
obtained upon addition of heparin Leo 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)
with excess concentrations under magnetic stirring at room
temperature. Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour. Heparin is totally or substantially totally
encapsulated in the nanoparticles. In other words, heparin is
substantially maintained within the intact nanoparticles during the
nanoparticle delivery phase orally. Thus, heparin does not cause
any significant effect until the nanoparticles dissociate or
biodegrade to release the core contents. Table 4 shows the
conditions of solution preparation and the average nanoparticle
size.
TABLE-US-00005 TABLE 4 Heparin Chitosan Particle Conditions conc.
@2 ml conc. @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
[0136] 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 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
[0137] In a further embodiment, a growth factor such as bFGF with
pharmaceutically effective amount is added to heparin Leo aqueous
solution before the pipetting step in Example No. 16. In our
laboratory, growth factors and proteins with pharmaceutically
effective amount 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 (VEGF 189), 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.
[0138] 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.
[0139] In one embodiment, it is disclosed that loading 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.
[0140] By modifying the chitosan structure to alter its charge
characteristics, such as grafting the chitosan with methyl, 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 or a polymer with a chemical formula:
##STR00003##
where R is .gtoreq.12
[0141] 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 pH 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 pH 2.5, preferably within at a pH as low as 1.0.
[0142] FIG. 16 shows an illustrative mechanism of nanoparticles
released from the enteric coating. FIG. 16(A) shows the phase of
nanoparticles in the gastric cavity, wherein the nanoparticles 82
are encapsulated within an initial enteric coating 81. FIG. 16(B)
shows a schematic of the coated nanoparticles during the phase of
entering small intestine, wherein the enteric coating starts to
dissolve 83 and a portion of nanoparticles 83 starts to release.
FIG. 16(C) shows the phase of nanoparticles in the intestinal
tract, wherein the nanoparticles open the tight junctions as
described above. 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.
[0143] It is known that Zn (zinc) is usually added in the
biosynthesis and storage of insulin. FIGS. 17 and 18 show a
schematic of insulin conjugated with a polyanionic compound (i.e.,
.gamma.-PGA in this case) via Zn and thus increase its loading
efficiency and loading content in the nanoparticles of the present
invention. It is further demonstrated that Zn may complex with the
histidine and glutamic acid residues in insulin to increase the
insulin stability and enhance controlled release capability or
sustained therapy. Some aspects of the invention relate to a
nanoparticle characterized by enhancing intestinal or brain blood
paracellular transport, the nanoparticle comprising 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 a stabilizer is added to complex the at
least one bioactive agent to the negatively charged third
component. In one embodiment, the stabilizer is zinc or
calcium.
[0144] One aspect describes a nanoparticle system encapsulating
abatacept for oral administration via intestinal absorption routes.
Orencia.RTM. (Abatacept) is an injectable, synthetic (man-made)
protein produced by recombinant DNA technology that is used for
treating rheumatoid arthritis. T-lymphocytes are important cells of
the immune system. Patients with rheumatoid arthritis have
increased numbers of T-lymphocytes within the joints that are
inflamed. The T-lymphocytes are activated, that is, they multiply
and release chemicals that promote the destruction of tissues
surrounding the joints and cause the signs and symptoms of
rheumatoid arthritis. Abatacept acts like an antibody and attaches
to a protein on the surface of T-lymphocytes. By attaching to the
protein, abatacept prevents the activation of the T-lymphocytes and
blocks both the production of new T-lymphocytes and the production
of the chemicals that destroy tissue and cause the symptoms and
signs of arthritis. Abatacept slows the damage to bones and
cartilage and relieves the symptoms and signs of arthritis.
Abatacept was approved by the FDA in December 2005. As typical with
injection, Abatacept is infused over 30 minutes. The initial dose
of abatacept is followed by a second dose two weeks later with
further doses every 4 weeks thereafter.
[0145] Monoclonal Antibody
[0146] Substances foreign to the body, such as disease-causing
bacteria and viruses and other infectious agents, known as
antigens, are recognized by the body's immune system as invaders.
Our natural defenses against these infectious agents are
antibodies, proteins that seek out the antigens and help destroy
them. Immunoglobulins are glycoproteins in the immunoglobulin
superfamily that function as antibodies. The terms antibody and
immunoglobulin are often used interchangeably. They are found in
the blood and tissue fluids, as well as many secretions. In
structure, they are globulins (in the .gamma.-region of protein
electrophoresis). They are synthesized and secreted by plasma cells
that are derived from the B cells of the immune system. B cells are
activated upon binding to their specific antigen and differentiate
into plasma cells. In some cases, the interaction of the B cell
with a T helper cell is also necessary. The nominal size of an
antibody is about 10 nm.
[0147] Humans have the ability to make antibodies able to recognize
(by binding to) virtually any antigenic determinant (i.e., epitope)
and to discriminate between even similar epitopes. An epitope is a
small piece of the antigen to which the antibody binds. Not only
does this provide the basis for protection against disease
organisms, but also it makes antibodies attractive candidates to
target other types of molecules found in the body, such as
receptors or other proteins present on the surface of normal cells
and molecules present uniquely on the surface of cancer cells. Thus
the remarkable specificity of antibodies makes them promising
agents for human therapy. It has been suggested to make an antibody
that will bind only to the cancer cells in a patient, couple a
cytotoxic agent (e.g. a strong radioactive isotope or
cancer-killing paclitaxel) to that antibody, and then give the
complex to the patient so it can seek out and destroy the cancer
cells (and no normal cells).
[0148] Monoclonal antibodies are widely used as diagnostic and
research reagents. Their introduction into human therapy has been
much slower. Reimbursement of self-administered biologics,
particularly the oral administration route, under Medicare Part D
will significantly improve access to biologics and expand the
market. In some in vivo applications, the antibody itself is
sufficient. Once bound to its target, it triggers the normal
effector mechanisms of the body, such as alerting other cells in
the immune system to the presence of the cancer cells. In other
cases, the monoclonal antibody is coupled to another molecule, for
example a fluorescent molecule to aid in imaging the target or a
strongly-radioactive atom, such as Iodine-131 to aid in killing the
target.
[0149] Some monoclonal antibodies that have been introduced into
human medicine include those that suppress the immune system, such
as: [0150] Muromonab-CD3 (OKT3) and two humanized anti-CD3
monoclonals. Bind to the CD3 molecule on the surface of T cells.
Used to prevent acute rejection of organ, e.g., kidney transplants.
The humanized versions show promise in inhibiting the autoimmune
destruction of beta cells in Type 1 diabetes mellitus. [0151]
Infliximab (Remicade.RTM.). Binds to tumor necrosis factor-alpha
(TNF-.alpha.). Shows promise against some inflammatory diseases
such as rheumatoid arthritis. [0152] Omalizumab (Xolair.RTM.).
Binds to IgE thus preventing IgE from binding to mast cells. Shows
promise against allergic asthma. [0153] Daclizumab (Zenapax.RTM.).
Binds to part of the IL-2 receptor produced at the surface of
activated T cells. Used to prevent acute rejection of transplanted
kidneys. Has also showed promise against T-cell lymphoma.
[0154] Some monoclonal antibodies that have been introduced into
human medicine include those that kill or inhibit malignant cells,
such as: [0155] Rituximab (Rituxan.RTM.). Binds to the CD20
molecule found on most B-cells and is used to treat B-cell
lymphomas. It shows efficacy for treating the patient population
that is refractory to treatment (either do not respond to therapy
or cannot tolerate therapy) with TNF inhibitors, such as
Adalimumab. [0156] Zevalin.RTM.. This is a monoclonal antibody
against the CD20 molecule on B cells (and lymphomas) conjugated to
either the radioactive isotope indium-111 (.sup.111In) or the
radioactive isotope yttrium-90 (.sup.90Y) [0157] Bexxar.RTM.
(tositumomab). This is a conjugate of a monoclonal antibody against
CD20 and the radioactive isotope iodine-131 (.sup.131I). It is
designed as a treatment for lymphoma. [0158] Herceptin.RTM.
(trastuzumab). Binds HER2, a receptor for epidermal growth factor
(EGF) that is found on some tumor cells (some breast cancers,
lymphomas). The only monoclonal so far that seems to be effective
against solid tumors. [0159] Erbitux.RTM. (cetuximab). Blocks HER1,
another epidermal growth factor (EGF) receptor. [0160]
Mylotarg.RTM.. A conjugate of a monoclonal antibody that binds
CD33, a cell-surface molecule expressed by the cancerous cells in
acute myelogenous leukemia (AML) but not found on the normal stem
cells needed to repopulate the bone marrow. calicheamicin, a
complex oligosaccharide that makes double-stranded breaks in DNA.
Mylotarg.RTM. is the first immunotoxin that shows promise in the
fight against cancer. [0161] LymphoCide. Binds to CD22, a molecule
found on some B-cell leukemias. [0162] Alemtuzumab
(MabCampath.RTM.). Binds to CD52, a molecule found on white blood
cells. Has produced complete remission of chronic lymphocytic
leukemia. [0163] Certolizumab pegol CDP870 (Cimzia.RTM.). Cimzia is
the first and only PEGylated Fab' fragment of a humanized anti-TNF
alpha antibody. The engineered Fab' fragment retains the biologic
potency of the original antibody. Cimzia has a high affinity for
human TNF alpha, selectively neutralizing the pathophysiological
effects of TNF alpha. Over the past decade, TNF has emerged as a
major target of basic research and clinical investigation. This
cytokine plays a key role in mediating pathological inflammation,
and excess TNF production has been directly implicated in a wide
variety of diseases. [0164] Lym-1 (Oncolym.RTM.). Binds to the
HLA-DR-encoded histocompatibility antigen that can be expressed at
high levels on lymphoma cells.
[0165] Some monoclonal antibodies that have been introduced into
human medicine include those that inhibit angiogenesis or perform
other function, such as: [0166] Vitaxin. Binds to a vascular
integrin (alpha-v/beta-3) found on the blood vessels of tumors but
not on the blood vessels supplying normal tissues. In Phase II
clinical trials, Vitaxin has shown some promise in shrinking solid
tumors without harmful side effects. [0167] Bevacizumab
(Avastin.RTM.). Binds to vascular endothelial growth factor (VEGF)
preventing it from binding to its receptor. Approved by the US FDA
in February 2004 for the treatment of colorectal cancers. [0168]
Abciximab (ReoPro.RTM.). Inhibits the clumping of platelets by
binding the receptors on their surface that normally are linked by
fibrinogen. Helpful in preventing reclogging of the coronary
arteries in patients who have undergone angioplasty.
[0169] In additional to the above monoclonal antibodies, other
approved monoclonal antibodies may include, but not limited to,
Basiliximab, Gemtuzumab ozogamicin, Ibritumomab, Palivizumab,
Eculizumab, Adalimumab (Humira.RTM.), and Efalizumab.
[0170] By way of illustration, Natalizumab is a monoclonal antibody
bioengineered from part of a mouse antibody to closely resemble a
human antibody. It is being marketed under the trade name
Tysabri.RTM.. The product is given intravenously once a month in a
physician's office for treating multiple sclerosis (MS) to reduce
the frequency of symptom flare-ups or exacerbations of the disease.
MS is a chronic, often disabling disease of the brain and spinal
cord.
[0171] By way of illustration, Rituximab targets a protein called
CD20, found on the surface of type B lymphocytes. These are one of
the main types of white blood cells, and the cell at fault in most
cases of non-Hodgkin's lymphoma. Although the drug attacks both
cancerous and non-cancerous type B cells, the body quickly replaces
them with healthy lymphocytes. Monoclonal antibodies have been
designed to recognize certain proteins found on the surface of some
cancer cells. Once the monoclonal antibody has recognized the
protein, it locks on to it and triggers the body's immune system to
attack the cancer cells, without affecting other cells in the body.
Sometimes it instructs the cells to destroy themselves.
[0172] One possible treatment for cancer involves monoclonal
antibodies (mAb) that bind only to cancer cell-specific antigens
and induce an immunological response against the target cancer
cell. Such mAb could also be modified for delivery of a toxin,
radioisotope, cytokine or other active conjugate; it is also
possible to design bispecific antibodies that can bind with their
Fab regions both to target antigen and to a conjugate or effector
cell. In fact, every intact antibody can bind to cell receptors or
other proteins with its Fc region.
[0173] Antibody Structure and Binding with Antigen
[0174] Immunoglobulins are heavy plasma proteins, often with added
sugar chains on N-terminal (all antibodies) and occasionally
O-terminal (IgA1 and IgD) amino acid residues. According to
differences in their heavy chain constant domains, immunoglobulins
are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and
IgE. Other immune cells collaborate with antibodies to eliminate
pathogens depending on which IgG, IgA, IgM, IgD, and IgE constant
binding domain receptors it can express on its surface.
[0175] The basic unit of each antibody is a monomer. An antibody
can be monomeric, dimeric, trimeric, tetrameric, pentameric, etc.
The monomer is a "Y"-shape molecule that consists of two identical
heavy chains and two identical light chains connected by disulfide
bonds. There are five types of heavy chain: .gamma., .delta.,
.alpha., .mu. and .epsilon.. They define classes of
immunoglobulins. Each heavy chain has a constant region, which is
the same by all immunoglobulins of the same class, and a variable
region, which differs between immunoglobulins of different B cells,
but is the same for all immunoglobulins produced by the same B
cell. Heavy chains .gamma., .alpha. and .delta. have the constant
region composed of three domains but have a hinge region; the
constant region of heavy chains .mu. and .epsilon. is composed of
four domains. The variable domain of any heavy chain is composed of
one domain. These domains are about 110 amino acids long. There are
also some amino acids between constant domains.
[0176] The "Y"-shaped monomer has two heavy and two light chains.
Together this gives six to eight constant domains and four variable
domains. Each half of the forked end of the "Y" is called an Fab
fragment. It is composed of one constant and one variable domain of
each the heavy and the light chain, which together shape the
antigen binding site at the amino terminal end of the monomer. The
two variable domains bind their specific antigens. The enzyme
papain cleaves a monomer into two Fab (fragment antigen binding)
fragments and an Fc (fragment crystallizable) fragment. The enzyme
pepsin cleaves below hinge region, so a Fab fragment and a Fc
fragment is formed. Together, the antibodies in an organism can
bind a wide variety of foreign antigens.
[0177] The antibodies have two primary functions: they bind
antigens and they combine with different immunoglobulin receptors
specific for them and exert effector functions. These receptors are
isotype-specific, which gives a great flexibility to the immune
system, because different situations require only certain immune
mechanisms to respond to antigens. By "Affinity" is herein defined
as the binding strength of the antibody to the antigen. By
"Avidity" is herein defined as the number of antigen binding
sites.
[0178] Antibodies exist freely in the bloodstream or bound to cell
membranes. They are part of the humoral immune system. Antibodies
exist in clonal lines that are specific to only one antigen, e.g.,
a virus hull protein. In binding to such antigens, they can cause
agglutination and precipitation of antibody-antigen products primed
for phagocytosis by macrophages and other cells, block viral
receptors, and stimulate other immune responses, such as the
complement pathway. Antibodies that recognize viruses can block
these directly by their sheer size. The virus will be unable to
dock to a cell and infect it, hindered by the antibody. They can
also agglutinate them so the phagocytes can capture them.
Antibodies that recognize bacteria mark them for ingestion by
phagocytes, a process called opsonization. Together with the plasma
component complement, antibodies can kill bacteria directly. They
neutralize toxins by binding with them.
[0179] In biochemistry, antibodies are used for immunological
identification of proteins, using the Western blot method. A
similar technique is used in ELISPOT and ELISA assays, in which
detection antibodies are used to detect cell secretions such as
cytokines or antibodies. Antibodies are also used to separate
proteins (and anything bound to them) from the other molecules in a
cell lysate. A Western blot (a.k.a immunoblot) is a method in
molecular biology/biochemistry/immunogenetics to detect protein in
a given sample of tissue homogenate or extract. It uses gel
electrophoresis to separate denatured proteins by mass. The
proteins are then transferred out of the gel and onto a membrane
(typically nitrocellulose), where they are "probed" using
antibodies specific to the protein. As a result, researchers can
examine the amount of protein in a given sample and compare levels
between several groups.
[0180] The Enzyme-Linked ImmunoSorbent Assay (ELISA for short) is a
biochemical technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample. It utilizes two
antibodies, one of which is specific to the antigen and the other
of which is coupled to an enzyme. This second antibody gives the
assay its "enzyme-linked" name, and will cause a chromogenic or
fluorogenic substrate to produce a signal. Because the ELISA can be
performed to evaluate either the presence of antigen or the
presence of antibody in a sample, it is a useful tool both for
determining serum antibody concentrations (such as with the Human
Immunodeficiency Virus, HIV test or West Nile Virus) and also for
detecting the presence of antigen. ELISPOT is an immunological
assay based on ELISA. Basically, the difference between the two is
that in ELISA, the substance containing the "unknown" is stuck at
the bottom of the well, whereas in ELISPOT the substance with the
"unknown" is placed in the well after the bottom of the well has
been coated with cytokine-specific antibody. ELISPOT is a method
for detecting cytokine production at the single cell level whereas
ELISA is a sensitive and specific method for quantification of
different cytokines.
[0181] Antibody detection kits, such as the ExtrAvidin.RTM.
Staining Kits (Sigma, St Louis, Mo.) are used to assay the presence
of monoclonal antibody in nanoparticles. These kits comprise
universal reagents for use with primary antibodies in
immunohistology, ELISA, and immunoblotting. ExtrAvidin is a unique
form of avidin that combines the high specificity and affinity of
avidin for biotin with low non-specific binding at physiological
pH. ExtrAvidin alkaline phosphatase and peroxidase enzymes thus
exhibit high sensitivity with low background. For example,
monoclonal anti-goat IgG antibodies in EXTRA-1 show no
cross-reactivity with human IgG. Further, affinity isolated
antibodies in EXTRA-2A and EXTRA-3A etc. have been adsorbed with
human IgG and IgM to minimize cross-reactivity. Biotinylated
antibodies contain a spacer which improves accessibility for the
ExtrAvidin conjugates.
Example No. 19
Nanoparticles with Monoclonal Antibody
[0182] One aspect describes nanoparticles for oral administration
in a patient comprising a positively charged shell substrate, a
negatively charged core substrate, and a bioactive agent
encapsulated within the nanoparticles, wherein the bioactive agent
is monoclonal antibody. In one embodiment, the bioactive agent is
mixed with, conjugated to, or coupled to the core substrate. In
another embodiment, the bioactive agent is totally encapsulated
within the nanoparticles.
[0183] Glycosaminoglycan (GAG), heparin, .alpha.-PGA, or
.gamma.-PGA is a negatively charged substrate that serves to bind
with positively charged chitosan substrate to form a nanoparticle.
Some aspects of the invention relate to GAG containing
nanoparticles for oral administration. In one embodiment, GAG
serves as at least a portion of the core substrate with chitosan
serves as at least a portion of the shell substrate, wherein GAG
conjugates at least one bioactive agent as disclosed herein, for
example, monoclonal antibodies. In preparation, nanoparticles were
obtained upon addition of aqueous solution (2 ml) of GAG and
monoclonal antibody "Bevacizumab", 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 CS
concentrations under magnetic stirring at room temperature.
[0184] Nanoparticles were collected by ultracentrifugation at
38,000 rpm for 1 hour and coded mAb-NP, which is formed of shell
substrate chitosan, core substrate GAG with encapsulated monoclonal
antibody. The monoclonal antibody is wholly or substantially
totally encapsulated in the nanoparticles. The nanoparticles have
an average diameter from about 50 nm to about 500 nm. In a
preferred embodiment, the nanoparticles are nanoshells having
biodegradable chitosan as a shell substrate. The nanoparticles may
further comprise a core substrate that mixes with the monoclonal
antibody or monoclonal antibody anti-cancer drug. In clinical
settings, the method of treating a tumor with a monoclonal antibody
anti-cancer drug that is released from the nanoparticles comprises
a step of binding the anti-cancer drug to a cell or tissue of the
patient, wherein the binding is by the formation of an
antigen-antibody complex or the formation of a ligand-receptor
complex. In one embodiment, the cell or tissue is cancerous. As
described above, the nanoparticles comprise a positively charged
shell substrate and a negatively charged core substrate, wherein
the nanoparticles have a surface charge from about +15 mV to about
+50 mV.
[0185] Avastin is a recombinant humanized monoclonal IgG1 antibody
that binds to and inhibits the biologic activity of human vascular
endothelial growth factor (VEGF) in in vitro and in vivo assay
systems, and is also sometimes described as a targeted therapy.
Avastin is a particular type of targeted therapy called
anti-angiogenic therapy that may interfere with the growth of new
blood vessels, which provide nutrients to the tumor. Avastin is the
first anti-angiogenic therapy in combination with first-line
chemotherapy proven to help many people with metastatic colorectal
cancer live longer. In clinical trials, patients taking Avastin in
combination with chemotherapy experienced many benefits compared
with those taking chemotherapy alone. In order to grow and spread,
tumors need a constant supply of oxygen and other nutrients. Tumors
get this supply by creating their own network of blood vessels.
This process is called angiogenesis.
[0186] To start angiogenesis, a tumor sends out signals to nearby
blood vessels. These signals cause new blood vessels to grow toward
the tumor. Once these new vessels reach the tumor, they provide the
supply of blood that provides oxygen and other nutrients to the
tumor. Avastin is thought to work by interfering with the signals
that cause angiogenesis. Avastin is given by infusion. Bevacizumab
is produced in a Chinese Hamster Ovary mammalian cell expression
system in a nutrient medium containing the antibiotic gentamicin
and has a molecular weight of approximately 149 kilodaltons.
AVASTIN is a clear to slightly opalescent, colorless to pale brown,
sterile, pH 6.2 solution for intravenous (IV) infusion.
[0187] The obtained mAb-NP in Example no. 18 is broken up by
extreme stirring/beating and the supernatant is assayed by ELISA to
confirm the presence of Bevacizumab antibody in the sample with
endothelial growth factor as target binding antigen.
[0188] Some aspects of the invention relate to a method of
delivering a monoclonal antibody protein to a target tissue or a
tumor of a patient, comprising the steps of: providing the
monoclonal antibody protein to the target tissue or the tumor,
wherein the monoclonal antibody protein is encapsulated within
nanoparticles; administering the nanoparticles to the patient
orally; and treating the target tissue or the tumor with the
monoclonal antibody protein that is released from the
nanoparticles. One preferred aspect of the invention relate to a
method of treating a target tissue or organ of a patient with a
monoclonal antibody, comprising the steps of: providing the
monoclonal antibody to the tissue or organ, wherein the monoclonal
antibody is encapsulated within nanoparticles; administering the
nanoparticles to the patient orally; and treating the target tissue
or organ with the monoclonal antibody that is sustained released
from the nanoparticles. In one embodiment, the monoclonal antibody
protein is an anti-cancer drug for the tumor. In another
embodiment, the monoclonal antibody is Adalimumab for treating
rheumatoid arthritis or psoriatic arthritis. In still another
embodiment, the monoclonal antibody protein is Bevacizumab for
treating tumor or cancer. FIG. 19 shows a schematic composition of
a nanoparticle with a shell substrate and a core substrate having a
monoclonal antibody. The method may further comprise a step of
delivering the nanoparticles to the target tissue or tumor through
intestinal absorption. In a preferred embodiment, the anti-cancer
monoclonal antibody is directed against tumor vasculature. Some
aspect provides that the target tumor is in an organ selected from
the group consisting of breast, lung, brain, liver, skin, kidney,
GI organ, prostate, bladder and gynecological organ.
Example No. 20
Nanoparticles with Monoclonal Antibody Adalimumab
[0189] Humira.RTM. (Adalimumab) is a recombinant human IgG1
monoclonal antibody specific for human tumor necrosis factor (TNF).
It consists of 1330 amino acids and has a molecular weight of
approximately 148 kilodaltons. The solution of Humira is clear and
colorless, with a pH of about 5.2 and contains 40 mg Adalimumab in
each 0.8 mL of single-use syringe. TNF is a naturally occurring
cytokine that is involved in normal inflammatory and immune
responses. Elevated levels of TNF are found in the synovial fluid
of rheumatoid arthritis and psoriatic arthritis patients and play
an important role in both the pathologic inflammation and the joint
destruction that are hallmarks of these diseases.
[0190] Clinically, after treatment with Humira, a rapid decrease in
levels of acute phase reactants of inflammation (C-reactive
protein, erythrocyte sedimentation rate, and serum cytokines IL-6)
was observed compared to baseline in patients with rheumatoid
arthritis. Serum levels of matrix metalloproteinases (MMP-1 and
MMP-3) that produce tissue remodeling responsible for cartilage
destruction were also decreased after Humira administration. Humira
is administered subcutaneously and/or intravenously. Adalimumab may
also be effective against plaque psoriasis, ankylosing spondylitis
or Crohn's disease. Crohn's disease is a chronic inflammatory
disease of the intestines. The disease, once it starts tends to
fluctuate between periods of inactivity (remission) and activity
(relapse).
[0191] In preparation of nanoparticles encapsulating Adalimumab,
the process comprises addition of aqueous solution (2 ml) of GAG
and monoclonal antibody "Adalimumab" by using a pipette (0.5-5 ml,
PLASTIBRAND.RTM., BrandTech Scientific Inc., Germany), into a
chitosan aqueous solution (pH 6.0, 10 ml) with excess CS
concentrations under magnetic stirring at room temperature.
Nanoparticles were collected by ultracentrifugation at 38,000 rpm
for 1 hour and were assayed by ELISA technique to confirm the
presence of Adalimumab monoclonal antibody in the nanoparticles
with target binding antigen. One aspect of the invention describes
a method of delivering a monoclonal antibody to a target tissue or
organ of a patient, comprising the steps of: providing the
monoclonal antibody to the tissue or organ, wherein the monoclonal
antibody is encapsulated within nanoparticles; administering the
nanoparticles to the patient orally; and treating the target tissue
or organ with the monoclonal antibody that is controlled released
from the nanoparticles. The method further comprises a step of
delivering the nanoparticles to the target tissue or organ through
intestinal absorption.
[0192] Complex of immunoglobulins with certain acidic
polysaccharides was demonstrated in the literature (Biochemistry
1997; 36:13187-13194) by the binding of the sulfated glycans
agaropectin or heparin with certain human IgG proteins. The major
binding force between heparin and the light chains of IgG is most
likely electrostatic. This electrostatic interaction is between the
cationic sites on certain IgG proteins and the anionic sulfate
residues of agaropectin or heparin. Prior research describes that
the interactions with multi-chain and single-chain rat skin heparin
are stable under physiological conditions and involve the Fab and,
more specifically, the Fv region of the IgG molecule (J. Exp. Med.
1981; 153:883-896). The characteristics of heparin-IgG interaction
resemble those of heparin with other plasma proteins, the
interactions of which have biological significance. Available
sulfated glycosaminoglycans include, but not limited to,
chondroitin 6-sulfate, keratin sulfate, heparin, and dermatan
sulfate. By ways of illustration, agaropectin found in agar
preparations is a polysaccharide. Some of its galactosyl units are
sulfated. Carrageenan has also sulfated side units for
complexion.
[0193] The following ELISA method has been adopted or slightly
modified for monoclonal antibody assay. The stock solutions
include: PBS (20 mM NaPi pH=7.5, 150 mM NaCl), PT (0.1% Tween 20 in
PBS), Blocking solution (3% BSA in PT; store at -20.degree. C.),
and Developing solution (10 mg o-phenylenediamine in 25 ml of a
buffer with pH=5.5 plus 12 .mu.l of 30% H.sub.2O.sub.2. This
solution is light sensitive and is prepared just before use).
[0194] The ELISA procedures include: [0195] 1. Coat the plate wells
with 50 .mu.l of antigen (0.2 .mu.g/ml in PBS). Incubate one hour
at room temperature; [0196] 2. Wash the plate (discard the well
solution, add 200 .mu.l of PT per well and wait about 2 min. Repeat
this cycle another 2 times and discard the late well solution);
[0197] 3. Block the plate wells with 150 .mu.l of blocking
solution. Incubate one hour at room temperature; [0198] 4. Discard
the blocking solution and add 50 .mu.l of the hybridoma
supernatants in different wells. Incubate 30 min at room
temperature; [0199] 5. Wash the plate; [0200] 6. Add 50 .mu.l of
the peroxidase-coupled antimouselgs antibody (1/5000 in blocking
solution). Incubate 30 min at room temperature; [0201] 7. Wash the
plate; [0202] 8. Incubate the plate wells with 100 .mu.l of
developing solution until color change is evident; [0203] 9. Stop
the reaction by adding 50 .mu.l of 2.5M H.sub.2SO.sub.4 per well;
and [0204] 10. Measure the absorbance at 492 nm.
[0205] Adalimumab is an injectable protein that blocks the
inflammatory effects of tumor necrosis factor alpha (TNF alpha) in
rheumatoid arthritis. Two other injectable drugs--Infliximab
(Remicade.RTM.) and etanercept (Enbrel.RTM.)--also block TNF alpha.
One aspect of the invention provides a nanoparticle comprising a
shell substrate of chitosan, a core substrate, and an encapsulated
bioactive agent that blocks TNF-alpha, wherein the bioactive agent
is selected from the group consisting of Adalimumab, Infliximab,
etanercept, and the like. Adalimumab was constructed from a fully
human monoclonal antibody, while infliximab is a mouse-human
chimeric antibody and etanercept is a TNF receptor-IgG fusion
protein. Inflammation is the body's reaction to injury and is a
necessary process for the repair of injury. TNF is a protein that
the body produces when there is inflammation. The TNF promotes
inflammation and the signs of inflammation, which, in the case of
arthritis, include fever as well as pain, tenderness, and swelling
of joints. The unchecked inflammation of rheumatoid arthritis
eventually leads to destruction of the joints. Adalimumab is a
synthetic (man-made) protein, similar to human protein, that binds
to TNF in the body and thereby blocks the effects of TNF. As a
result, inflammation and fever as well as the pain, tenderness and
swelling of joints are reduced in patients with rheumatoid
arthritis. In addition, the progressive destruction of the joints
is slowed or prevented. Adalimumab was approved by the FDA in
December 2002.
[0206] Statin
[0207] The statins (or HMG-CoA reductase inhibitors) form a class
of hypolipidemic agents, used as pharmaceuticals to lower
cholesterol levels in people at risk for cardiovascular disease
because of hypercholesterolemia. The first statin (Lovastatin) to
be marketed in shown below.
##STR00004##
[0208] The statins include, in alphabetical order (brand names vary
in different countries): Atorvastatin (Lipitor; disclosed in U.S.
Pat. No. 4,681,893), Cerivastatin (Lipobay, Baycol; disclosed in
U.S. Pat. No. 5,006,530), Fluvastatin (Lescol; disclosed in U.S.
Pat. No. 4,739,073), Lovastatin (Mevacor, Altocor; disclosed in
U.S. Pat. No. 4,231,938), Mevastatin (naturally-occurring compound,
found in red yeast rice), Pravastatin (Pravachol, Selektine,
Lipostat; disclosed in U.S. Pat. No. 4,346,227), Rosuvastatin
(Crestor; disclosed in U.S. Pat. No. 5,260,440), Simvastatin
(Zocor, Lipex; disclosed in U.S. Pat. No. 4,444,784), and
Pitavastatin (disclosed in U.S. Pat. No. 5,011,930). Their chemical
formulas are listed in U.S. Pat. No. 6,777,552 B2, entire contents
of which are incorporated herein by reference. LDL-lowering potency
varies between agents. Among the above-identified statins,
pravastatin, fluvastatin, cerivastatin, atovastatin, rosuvastatin,
and pitavastatin are in acidic forms (that is, having carboxylic
units) and can become sodium or calcium salt forms. One aspect of
the invention relates to a nanoparticle comprising positively
charged shell substrate chitosan and negatively charged core
substrate statins, such as the ones with carboxylic units. In one
embodiment, the statins are complexed with chitosan, low MW
chitosan, or chitosan derivatives.
[0209] Statins act by competitively inhibiting HMG-CoA reductase,
an enzyme of the HMG-CoA reductase pathway, the body's metabolic
pathway for the synthesis of cholesterol. Although statins inhibit
endogenous cholesterol synthesis, their action goes further than
that. By reducing intracellular cholesterol levels, they cause
liver cells to upregulate expression of the LDL receptor, leading
to increased clearance of low-density lipoprotein from the
bloodstream. Due to its sustained release characteristics of the
nanoparticle system of the present invention (as evidenced in FIG.
15), one aspect provides a nanoparticle that encapsulates at least
one statin adapted for sustained release and sustained inhibition
of the HMG-CoA reductase pathway (Berg, J. M., J. L. Tymoczko, and
L. Stryer, Biochemistry. 5th ed. 2002, New York: W.H. Freeman.
xxxviii, 974). Statins is one drug that targets the HMG-CoA
reductase pathway (used for elevated cholesterol levels). Another
bioactive agent for targeting the HMG-CoA reductase pathways is
Bisphosphonates (used for osteoporosis).
[0210] Statins exhibit action beyond lipid-lowering activity in the
prevention of atherosclerosis. Researchers believe that statins
prevent cardiovascular disease via four proposed mechanisms:
improving endothelial function, modulating inflammatory responses,
maintaining plaque stability, and preventing thrombus formation. It
was recently reported that statin therapy could significantly
reduce morbidity and mortality in diabetics. The decision to treat
is based on vascular risk and not initial cholesterol levels
(MRC/BHF Heart Protection Study of cholesterol-lowering with
simvastatin in 5963 people with diabetes: a randomized
placebo-controlled trial. Lancet 2003 361: 2005-2016). Therefore,
it is contemplated that insulin and statin may be co-encapsulated
in nanoparticles system of the present invention.
[0211] Very high-intensity statin therapy can result in significant
regression of coronary atherosclerosis. A landmark two-year study
demonstrated that CRESTOR.TM. (rosuvastatin) reversed plaque
build-up in the arteries of patients with evidence of coronary
artery disease. Researchers at the Cleveland Clinic treated 507
patients with 40 mg/d for 24 months and use intravascular
ultrasound before and after treatment to measure changes in
atheroma volume. This is the first time a statin has demonstrated
regression of atherosclerosis in a major clinical study. Data
presented from ASTEROID, at the 55th Annual Scientific Session of
the American College of Cardiology (ACC) in 2006 and published in
the April 5.sup.th issue of the Journal of the American Medical
Association show that plaque build-up in patients' arteries was
reduced by between seven and nine percent. These significant
changes were achieved with CRESTOR and were associated with
significant reductions in LDL-C or `bad` cholesterol (53 percent,
p<0.001) and increases in HDL-C or `good` cholesterol (15
percent, p<0.001).
[0212] With evidence from observational studies, as well as
retrospective analyses of previous trials, there had been
speculation that statins might play a role in reducing the
recurrence of ventricular arrhythmias. New results from a
prospective, randomized, placebo-controlled clinical trial support
that hypothesis, with the evidence suggesting that statins appear
to have antiarrhythmic effects (De Sutter J et al. "Intensive
lipid-lowering therapy and ventricular arrhythmias in patients with
coronary artery disease and internal cardioverter defibrillators"
Heart Rhythm Society 2006 Scientific Sessions; May 17-20, 2006;
Boston, Mass.). Intensive lipid-lowering therapy, using
atorvastatin 80 mg, is an effective and safe way to reduce the
recurrence of ventricular arrhythmias in patients with coronary
artery disease and ICD implants. It was reported that treatment
with atorvastatin resulted in a statistically significant 59%
reduction in the number of days with an appropriate ICD
intervention and high-dose statin therapy in these patients was
safe and well tolerated, with no statistically significant
treatment-related adverse events observed.
[0213] Statins do not increase the risk for breast cancer and
hydrophobic statins may decrease the risk, according to the results
of an analysis of the Women's Health Initiative (WHI) study (J Natl
Cancer Inst. 2006; 98:700-707). To evaluate associations between
type of statin used, potency, duration of use, and risk for
invasive breast cancer, the investigators examined data for 156,351
postmenopausal women enrolled in the WHI, of whom 11,710 (7.5%)
used statins. During an average follow-up of 6.7 years, 4383
invasive breast cancers were identified by review of medical
records and pathology reports. Breast cancer incidence was 4.09 per
1000 person-years for statin users and 4.28 per 1000 person-years
for nonusers. Duration of statin use did not affect risk.
Hydrophobic statins, such as simvastatin, lovastatin, and
fluvastatin, were used by 8106 women, and their use was associated
with an 18% lower incidence of breast cancer. Use of other statins,
such as pravastatin and atorvastatin, or nonstatin lipid-lowering
agents, was not associated with breast cancer incidence. A number
of different mechanisms have been identified by which hydrophobic
statins might inhibit the growth of cancer. In this large
population of postmenopausal women with well-characterized breast
cancer risk factors, when all statins were considered together as a
class, no statistically significant association with breast cancer
incidence was seen.
Example No. 21
Nanoparticles with Encapsulated Statin
[0214] FIG. 19 shows a schematic composition of a nanoparticle with
a shell substrate and a core substrate having a statin (HMG-CoA
reductase inhibitor). In product formulation, nanoparticles were
obtained upon addition of statin (in one example for illustration,
atorvastatin) 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) with excess CS
concentrations under magnetic stirring at room temperature.
Nanoparticles were collected by ultracentrifugation at 30,000 rpm
for 1 hour. Nanoparticles comprise positively charged shell
substrate chitosan and negatively charged core substrate statins.
Statin is totally or substantially totally encapsulated in the
nanoparticles. In other words, statin component is substantially
maintained within the intact nanoparticles during the nanoparticle
delivery phase orally. Thus, statin does not cause any significant
effect until the nanoparticles dissociate or biodegrade to release
the core contents in a sustained release manner.
[0215] Some aspects of the invention relate to a method of
delivering an HMG-CoA reductase inhibitor to blood circulation in a
patient, comprising: (a) providing nanoparticles that encapsulate
the HMG-CoA reductase inhibitor, wherein the nanoparticles are
biodegradable; (b) administering the nanoparticles orally toward an
intestine of the patient; (c) urging the nanoparticles to pass
through an epithelial barrier of the intestine; and (d) releasing
the HMG-CoA reductase inhibitor into the blood circulation in a
sustained manner. The sustained release of a bioactive agent from a
chitosan-shelled nanoparticle has been demonstrated in FIG. 15,
with reference to that via subcutaneous injection. In one aspect of
the invention, the method further comprises a step of enhancing the
chitosan-shelled nanoparticles to adhere to a mucosal surface of
the intestine for paracellular transport. In one embodiment, the
HMG-CoA reductase inhibitor is a statin or bisphosphonates.
[0216] 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.
* * * * *