U.S. patent application number 17/093186 was filed with the patent office on 2021-05-27 for cationic nanostructures for intra-cartilage delivery of therapeutics and contrast agents.
The applicant listed for this patent is Northeastern University. Invention is credited to Ambika Bajpayee, Tengfei He, Armin Vedadghavami, Chenchen Zhang.
Application Number | 20210154307 17/093186 |
Document ID | / |
Family ID | 1000005418185 |
Filed Date | 2021-05-27 |
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United States Patent
Application |
20210154307 |
Kind Code |
A1 |
Bajpayee; Ambika ; et
al. |
May 27, 2021 |
CATIONIC NANOSTRUCTURES FOR INTRA-CARTILAGE DELIVERY OF
THERAPEUTICS AND CONTRAST AGENTS
Abstract
The present invention provides a platform for the delivery of
small molecule drugs or contrast agents to joints and other soft
tissues. The platform enables penetration of the drug through the
full thickness of cartilage and long intra-cartilage residence time
by leveraging electrostatic interactions between the cationic
platform and the anionic cartilage matrix. Described herein are
compounds and complexes that fit this platform. Also provided are
methods of treating a joint disease with the compounds and
complexes of the invention, and methods of imaging joints and other
soft tissue.
Inventors: |
Bajpayee; Ambika; (Boston,
MA) ; Zhang; Chenchen; (Boston, MA) ;
Vedadghavami; Armin; (Boston, MA) ; He; Tengfei;
(Malden, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northeastern University |
Boston |
MA |
US |
|
|
Family ID: |
1000005418185 |
Appl. No.: |
17/093186 |
Filed: |
November 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62932232 |
Nov 7, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/22 20130101;
A61K 9/0019 20130101; A61K 38/18 20130101; A61K 47/60 20170801;
A61K 47/645 20170801; A61K 31/573 20130101; B82Y 5/00 20130101;
A61K 49/0438 20130101 |
International
Class: |
A61K 47/60 20060101
A61K047/60; A61K 38/18 20060101 A61K038/18; A61K 31/573 20060101
A61K031/573; A61K 47/64 20060101 A61K047/64; A61K 9/00 20060101
A61K009/00; A61K 47/22 20060101 A61K047/22; A61K 49/04 20060101
A61K049/04 |
Goverment Interests
GOVERNMENT SUPPORT CLAUSE
[0002] This invention was made with government support under Grant
No. R03EB025903 awarded by the National Institutes of Health, and
Grant No. W81XWH-17-1-0085 awarded by the Department of Defense.
The government has certain rights in the invention.
Claims
1. A compound, comprising: a residue of biotin; a multi-arm
polymeric scaffold; one or more hydrolyzable linking moieties; and
one or more residues of an active pharmaceutical ingredient;
wherein the residue of biotin is covalently attached to the
multi-arm polymeric scaffold; each of the one or more hydrolyzable
linking moieties are covalently attached to the multi-arm polymeric
scaffold; and each of the one or more residues of the active
pharmaceutical ingredient are covalently attached to each of the
one or more hydrolyzable linking moieties.
2. (canceled)
3. (canceled)
4. The compound of claim 1, wherein one arm of the multi-arm
polymeric scaffold is covalently attached to the residue of biotin,
and each of the remaining arms of the multi-arm polymeric scaffold
is attached to a hydrolyzable linking moiety, and each hydrolyzable
linking moiety is attached to a residue of an active pharmaceutical
ingredient.
5.-7. (canceled)
8. The compound of claim 1, wherein the multi-arm polymeric
scaffold comprises 8 arms.
9.-12. (canceled)
13. The compound of claim 1, wherein each of the one of more
hydrolyzable linking moieties is selected from the group consisting
of ##STR00023##
14.-16. (canceled)
17. The compound of claim 1, wherein the active pharmaceutical
ingredient is a glucocorticoid or pharmaceutically acceptable salt
thereof.
18.-20. (canceled)
21. The compound of claim 1, wherein: the multi-arm polymeric
scaffold comprises an 8-arm polyethylene glycol scaffold; each of
the one of more hydrolyzable linking moieties is selected from the
group consisting of ##STR00024## and the active pharmaceutical
ingredient is dexamethasone.
22. The compound of claim 1, having the structure: ##STR00025##
wherein X is a hydrolyzable linking moiety selected from the group
consisting of ##STR00026## and n is an integer from 1 to 500,000;
or a pharmaceutically acceptable salt thereof.
23. (canceled)
24. A complex, comprising avidin and one or more compounds of claim
1.
25. The complex of claim 24, wherein the one or more compounds are
bound to avidin through electrostatic interactions.
26. The complex of claim 24, wherein avidin and the one or more
compounds are in a molar ratio of 1:4; and all four biotin-binding
sites of avidin are bound to one of the one or more compounds.
27.-34. (canceled)
35. A method of treating a joint disease in a subject in need
thereof, comprising administering to the subject a compound of
claim 1.
36.-39. (canceled)
40. A method of preventing glycosaminoglycan (GAG) loss in a
subject in need thereof, comprising administering to the subject a
complex of claim 24.
41. (canceled)
42. A compound, comprising: a residue of biotin; a multi-arm
polymeric scaffold; and one or more residues of a contrast agent;
wherein the residue of biotin is covalently attached to the
multi-arm polymeric scaffold; and each of the one or more residues
of a contrast agent are covalently attached to the multi-arm
polymeric scaffold.
43.-48. (canceled)
49. The compound of claim 42, wherein the multi-arm polymeric
scaffold comprises 8 arms.
50.-53. (canceled)
54. The compound of claim 42, wherein the contrast agent is
selected from the group consisting of diatrizoate, metrizoate,
iothalamate, and ioxaglate.
55. (canceled)
56. The compound of claim 42, having the structure: ##STR00027## n
is an integer from 1 to 500,000; or a pharmaceutically acceptable
salt thereof.
57. A complex, comprising avidin and one or more compounds of claim
42.
58.-63. (canceled)
64. A method of diagnosing a joint disease, comprising
administering to a subject a complex of claim 57; imaging the
joint; and assessing the resultant images to determine whether the
subject is suffering from a joint disease.
65.-68. (canceled)
69. A method of imaging soft tissue, comprising administering to a
subject a complex of claim 57; and imaging the soft tissue.
70.-71. (canceled)
72. A method of delivering a contrast agent or an active
pharmaceutical ingredient to a negatively charged tissue in a
subject, comprising administering to the subject a complex; wherein
the complex comprises a residue of a contrast agent or an active
pharmaceutical ingredient and a cationic peptide, wherein the
peptide comprises from 2 to 40 amino acid residues, and the net
charge of the peptide is from +7 to +20 inclusive; and the residue
of the contrast agent or the active pharmaceutical ingredient is
covalently bonded to the peptide.
73.-84. (canceled)
Description
RELATED APPLICATION
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 62/932,232, filed Nov. 7,
2019.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 27, 2021, is named NEX-06201 SL.txt and is 2,013 bytes in
size.
BACKGROUND
[0004] Osteoarthritis (OA) is a common chronic inflammatory disease
of the whole joint affecting knees, hips, fingers, and low spinal
regions, and is one of the most disabling diseases in developed
countries with an estimated social cost between 1 and 2.5% of gross
domestic product. Traumatic joint injuries lead to development of
post-traumatic OA (PTOA) within 10 years of injury in a majority of
cases. Following a joint injury, there is an immediate increase in
synovial fluid levels of inflammatory cytokines (e.g. interleukin-1
(IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha
(TNF.alpha.)) which diffuse into cartilage and rapidly initiate
proteolysis and matrix loss. While several drugs have known
potential to inhibit OA, none have yet translated to clinical
practice as they suffer from poor cartilage targeting and
off-target side effects.
[0005] Relatedly, early diagnosis of degenerative joint diseases
like osteoarthritis (OA) is critical as there is only a narrow
time-window during which therapeutic intervention can reverse
disease progression. While computed tomography (CT) can diagnose
changes in subchondral bone, it is not clinically viable for
imaging joint soft tissues like cartilage that exhibit early
degenerative changes associated with OA onset. CT may be developed
for soft tissue imaging by using radio-opaque contrast agents
injected into the joint, as long as they are safe and the time to
produce sufficient CT attenuation is short enough to be clinically
viable.
[0006] Currently, intra-articular (IA) injections to affected
joints are the primary route for directly delivering pain and
inflammation relievers, however, a majority of drug is rapidly
cleared from the joint space, therefore requiring multiple
injections of high drug doses which causes systemic toxicity. This
problem is further aggravated by the complex architecture of
avascular cartilage containing a dense meshwork of collagen,
interspersed with a high density of negatively charged
aggrecan-associated glycosaminoglycans (GAGs) which hinder drug
penetration. Breakdown of articular cartilage triggers production
of various inflammatory cytokines which disturb chondrocyte
homeostasis, causing these cells to produce more matrix degrading
agents than matrix generating agents. It is therefore critical for
drugs to reach their cell and matrix targets sites, requiring novel
strategies to enhance localization of OA drugs to target
chondrocytes. Currently there are no delivery systems that can (i)
locally and safely target cartilage and enable drugs to penetrate
through the full depth of tissue to reach chondrocytes and matrix
targets, (ii) bind within cartilage to prevent their diffusion back
out to the synovial fluid, and (iii) provide sustained drug release
over several days to weeks. None of the drug delivery systems
proposed thus far (e.g. drug-encapsulating micelles, liposomes,
polymeric particles, aggregating hydrogels, etc.) are able to
penetrate the cartilage or bind with its cell/matrix targets.
[0007] Similarly, contrast agents like ioxaglate (IOX) are anionic
and thus repelled by the negatively charged cartilage matrix
containing a high density of GAGs. This hinders the intra-tissue
penetration and partitioning of the contrast agents, and results in
poor CT attenuation. These contrast agents are also rapidly cleared
from the joint space, thus exhibiting short intra-tissue residence
time. Higher doses of contrast agents are therefore required to
enhance flux and achieve intra-cartilage concentrations sufficient
for CT imaging. Such high dosages result in both local and systemic
toxicity.
[0008] The high negative fixed charge density (FCD) of cartilage
resulting from the high density of negatively charged GAG chains
provides a unique opportunity to use electrostatic interactions for
enhancing transport, uptake and retention of cationic drug
carriers. Using short length cationic peptide carrier motifs, it
was shown recently that there exists an optimal net positive charge
to deliver a drug of given size to a tissue of known FCD that will
result in rapid penetration through the full thickness of cartilage
before a majority of it is cleared from the joint space, providing
the highest intra-cartilage uptake and long-term retention [1].
Optimal net positive charge on the carrier is chosen to enable weak
and reversible binding with the intra-tissue negatively charged
groups such that the drug and its carrier can penetrate through the
full tissue thickness and not get stuck in the tissue's superficial
zones. Despite weak binding, the high negative FCD of aggrecan
associated GAGs inside cartilage greatly increases the residence
time of optimally charged cationic drug carriers. Similarly, the
cationic glycoprotein, Avidin, due to its optimal net size (<10
nm hydrodynamic diameter) and charge (between +6 and +20) was shown
to penetrate through full thickness of rabbit cartilage following
IA injection [2], resulting in a high intra-cartilage uptake ratio
of 180 (implying 180.times. higher concentration of Avidin inside
cartilage than surrounding fluid at equilibration). Further, Avidin
was found to be present through the full thickness of cartilage two
weeks following its IA administration in a rabbit anterior cruciate
ligament transection (ACLT) model of PTOA [3]. Avidin was
covalently conjugated with 4 moles of Dexamethasone (Av-Dex) using
its four biotin binding sites [4] and administered in a single low
dose IA injection one week following ACLT in a rabbit model. Av-Dex
suppressed injury induced joint inflammation, synovitis, incidence
of osteophyte formation and restored trabecular properties at 3
weeks significantly greater than free Dex. However, to deliver even
a single low dose of 0.5 mg Dex, a high dose of 20 mg Avidin was
required due to the low drug loading content of the conjugate. This
design results in enhanced GAG loss from cartilage. Moreover, a
high dose of Avidin can reduce intra-tissue osmotic swelling
pressures owing to its cationic charge leading to decreased water
content and potential loss of proteoglycans. By contrast, Avidin
doses less than 100 .mu.M have been shown to not affect GAG loss,
chondrocyte viability or biosynthesis rates of proteins and GAGs in
bovine cartilage explants.
[0009] Therefore, there exists a need to develop a conjugate system
with an increased drug loading content for delivering effective
doses of drugs or contrast agents, which leverages the cartilage
homing property of Avidin, while decreasing the overall dosage of
Avidin, thus minimizing the GAG loss caused by Avidin.
SUMMARY OF INVENTION
[0010] In certain aspects, the present invention provides a
compound, comprising: [0011] a residue of biotin; [0012] a
multi-arm polymeric scaffold; [0013] one or more hydrolyzable
linking moieties; and one or more residues of an active
pharmaceutical ingredient; [0014] wherein the residue of biotin is
covalently attached to the multi-arm polymeric scaffold; each of
the one or more hydrolyzable linking moieties are covalently
attached to the multi-arm polymeric scaffold; and each of the one
or more residues of the active pharmaceutical ingredient are
covalently attached to each of the one or more hydrolyzable linking
moieties.
[0015] In other aspects, the present invention provides a complex,
comprising avidin and one or more compounds described herein.
[0016] The present invention also provides methods of treating a
joint disease in a subject, the method comprising administering to
the subject a compound or complex described herein.
[0017] The present invention also provides methods of preventing
glycosaminoglycan (GAG) loss in a subject in need thereof,
comprising administering to the subject a compound or complex
described herein.
[0018] In further aspects, the present invention provides a
compound comprising: [0019] a residue of biotin; [0020] a multi-arm
polymeric scaffold; and one or more residues of a contrast agent;
[0021] wherein the residue of biotin is covalently attached to the
multi-arm polymeric scaffold; and each of the one or more residues
of a contrast agent are covalently attached to the multi-arm
polymeric scaffold.
[0022] In other aspects, the present invention provides a complex,
comprising avidin and one or more such compounds described
herein.
[0023] Also provided herein is a method of diagnosing a joint
disease, comprising administering to a subject a compound or
complex described herein.
[0024] Also provides are methods of imaging soft tissue, comprising
administering to a subject a compound or complex of the invention,
and imaging the soft tissue.
[0025] Also provided herein are methods of delivering a contrast
agent or an active pharmaceutical ingredient to a negatively
charged tissue in a subject, comprising administering to the
subject a complex; [0026] wherein the complex comprises a residue
of a contrast agent or an active pharmaceutical ingredient and a
cationic peptide, wherein the peptide comprises from 2 to 40 amino
acid residues, and the net charge of the peptide is from +7 to +20
inclusive; and the residue of the contrast agent or the active
pharmaceutical ingredient is covalently bonded to the peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 depicts a schematic of charge-based intra-cartilage
drug delivery of the nano-construct multi-arm Avidin conjugated
with a small molecule drug, Dexamethasone (mAv-Dex) by using
hydrolysable ester linkers derived from succinic, glutaric and
phthalic anhydrides (SA, GA, PA) in 2:1:1 molar ratio enabling
tunable (and long) drug release half-lives. Following its
intra-articular administration, mAv-Dex can rapidly penetrate
through the full thickness of negatively charged cartilage in high
concentrations due to electrostatic interactions thereby creating
an intra-cartilage drug depot. The optimal net positive charge of
mAv enables its rapid and high intra-cartilage uptake and long-term
retention via weak-reversible binding with negatively charged
aggrecans. Therapeutic doses of drug (Dex) is then released via
hydrolysis from mAv over several days, which can reach the
chondrocytes to bind with its glucocorticoid receptors triggering
downstream signaling pathways and suppressing OA associated
catabolic activity. This intra-cartilage depot drug delivery
platform can be used to deliver a variety of drugs or combination
of drugs and enable OA treatment with only a one shot injection of
low drug doses thereby eliminating toxicity issues associated with
multiple injections of high drug doses that are currently needed to
maintain sustained drug doses within the joint.
[0028] FIG. 2A depicts the characterization of multi-arm Avidin
(mAv) synthesis. Schematic of HABA dye assay: HABA binds with
Avidin resulting in a high absorbance value but is competitively
displaced by biotin or biotinylated PEG reducing the absorbance
value. (Bottom) Titration curve of biotinylated PEG with
HABA-Avidin mixture. Absorbance value dropped with increasing
biotinylated PEG:HABA-Avidin molar ratio, and a plateau was
achieved after 4:1 molar ratio confirming that all four binding
sites of Avidin (Av) were occupied by PEGs to form 1:4 mAv
configuration.
[0029] FIG. 2B depicts the characterization of multi-arm Avidin
(mAv) synthesis. Native PAGE gel (7.5%) of Av, PEG, 1:2 mAv and 1:4
mAv under reverse polarity stained with (left) iodine for PEGs and
with (right) Coomassie Brilliant Blue R-250 for protein.
[0030] FIG. 2C depicts the characterization of multi-arm Avidin
(mAv) synthesis. UPLC analysis of (left) native Av with peak `a` at
6.29 min and (right) of 1:4 mAv formulation containing a majority
of mAv with 4 PEGs (peak `b` at 4.38 min) and a secondary
population of mAv with 2 PEGs (peak `c` at 5.33 min).
[0031] FIG. 3A shows the transport properties of mAv
nano-constructs in cartilage. Non-equilibrium diffusion curve of
1:4 mAv in cartilage showing normalized downstream concentration
(C.sub.D) to upstream concentration (Cu) versus time. .tau..sub.Lag
was used to estimate effective diffusivity, D.sub.EFF, in presence
of binding interactions within the cartilage. L refers to cartilage
thickness.
[0032] FIG. 3B shows intra-cartilage cartilage equilibrium uptake
of Avidin (Av), 1:2 mAv and 1:4 mAv after 24 h in healthy and 50%
GAG depleted arthritic cartilage (* vs respective condition for Av,
# vs respective healthy condition, p<0.05).
[0033] FIG. 3C shows the % retention of Av, 1:2 and 1:4 mAv inside
healthy cartilage following desorption in 1.times.PBS and
10.times.PBS over 7 days.
[0034] FIG. 3D shows confocal microscopy images demonstrating full
thickness intra-cartilage penetration of dual labeled 1:4 mAv from
superficial zone (SZ) to deep zone (DZ) of healthy and 50% GAG
depleted cartilage. Red channel shows Texas Red labeled Avidin and
green channel shows presence of FITC labeled PEGs in mAv.
[0035] FIGS. 4A-4D show a reaction scheme, release profile and
cytotoxicity of PEG-Dex compounds.
[0036] FIG. 4A shows a schematic of Dex conjugation with 8-arm
PEG-amine using cross linkers (R: SA, GA and PA) to form ester
linkers with varying carbon spacer lengths (X: X.sub.1, X.sub.2 and
X.sub.3).
[0037] FIG. 4B shows the corresponding Dex release rates at
37.degree. C., pH 7.4 in PBS. Controlled release PEG-Dex (2:1:1)
represents Dex conjugated using combination of ester linkers
synthesized from SA, GA and PA in 2:1:1 molar ratio of Dex.
[0038] FIG. 4C shows a mechanism of fast hydrolysis of PEG-Dex-SA
(5) in PBS (pH 7.4). Carbonyl in amide bond, an electron
withdrawing group (EWG), withdraws electrons from methylene and
ester bonds (inductive effect) thereby decreasing ester bond's
electron density and making it more electrophilic (.delta..sup.+)
and reactive to nucleophilic attack from hydroxide ion (.sup.-OH)
causing faster hydrolysis of the ester bond. Repulsion between
hydrophilic PEG and hydrophobic Dex further strains the ester
linker making it unstable. When X.sub.1 is replaced by X.sub.2 or
X.sub.3, the carbon spacer length is increased that weakens the
inductive effect of carbonyl and donates more electrons to
stabilize the ester bond. This also reduces the repulsive effects
between PEG and Dex.
[0039] FIG. 4D is a bar graph showing results of a cytotoxicity
test: % cell viability in cartilage explants following 48 h
treatment with PEG-Dex-SA, PEG-Dex-GA, and PEG-Dex-PA.
[0040] FIG. 5A shows the effectiveness of single dose mAv-Dex
treatment in suppressing IL-1.alpha. induced GAG loss and
chondrocyte death. % cumulative GAG loss over 16 days: IL-1.alpha.
treated cartilage explants treated a single or continuous (C) dose
of 100 nM free Dex.
[0041] FIG. 5B shows the effectiveness of single dose mAv-Dex
treatment in suppressing IL-1.alpha. induced GAG loss and
chondrocyte death. % cumulative GAG loss over 16 days: IL-1.alpha.
treated cartilage explants treated single doses of 10 .mu.M free
Dex, mAv-Dex or mAv-Dex (2:1:1). A concentration of 10 .mu.M mAv
alone in absence of IL-1.alpha. was also tested and compared with
the control condition (* vs control, # vs IL-1.alpha., $ vs single
dose Dex condition, A vs mAv-Dex, p<0.05. Statistical markers
are color coordinated with the curves. All data enclosed within
statistical markers are significant.)
[0042] FIG. 5C shows chondrocyte viability after 16 days of
culture. Green indicates viable cells and red indicates non-viable
cells. Scale bar=200 .mu.m.
[0043] FIG. 6A depicts the effect of mAv-Dex on nitrite release in
IL-1.alpha. treated cartilage. FIG. 6A shows nitrite released to
the medium per mg cartilage tissue on Day 2 and Day 8.
[0044] FIG. 6B depicts the effect of mAv-Dex on cell metabolism in
IL-1.alpha. treated cartilage. FIG. 6B shows chondrocyte metabolism
measured as relative fluorescence units (RFU) using alamarBlue
assay on Day 16. (* vs untreated, # vs IL-1.alpha., $ vs single
dose Dex of same concentration, p<0.05. Statistical markers are
color coordinated with the bars.)
[0045] FIG. 7 shows histological and immunohistochemical analysis
of cartilage. Cartilage explants stained with Safranin-O and Fast
Green (for GAG) or immunostained for collagen type II after a
16-day culture period.
[0046] FIG. 8A depicts the structure of cartilage tissue showing
increasing concentration of negatively charged GAGs from
superficial zone (SZ) to deep zone (DZ) of tissue giving it a high
negative fixed charge density (FCD).
[0047] FIG. 8B shows cationic contrast agent, mAv-IOX, due to its
optimal net charge can rapidly penetrate through the full thickness
of cartilage in high concentrations owing to high Donnan
partitioning factor (K) that increases its concentration at tissue
interface by KC. On the contrary, transport of anionic IOX is
hindered by negatively charged cartilage whose concentration at
cartilage interface partitions down by K'C.
[0048] FIG. 8C shows mAv-IOX results in high CT signal inside
cartilage at a low concentration of 0.5 mg I/mL while anionic IOX
cannot.
[0049] FIG. 9 is a 2D color map of cartilage treated with 0.5 mg
I/mL IOX, 16 mg I/mL IOX and 0.5 mg I/mL mAv-IOX. 0.5 mg I/mL
mAv-IOX produces high CECT attenuation than 0.5 I/mL IOX and shows
similar CECT attenuation to 16 mg I/mL IOX.
[0050] FIG. 10A contains graphs showing the correlation between
CECT attenuation and GAG content of cartilage using 16 mg I/mL
IOX.
[0051] FIG. 10B contains graphs showing the correlation between
CECT attenuation and GAG content of cartilage using 0.5 mg I/mL
mAv-IOX.
[0052] FIG. 11A shows a graph confirmation of PEG.
[0053] FIG. 11B shows a graph confirmation of biotinylated PEG
using MALDI-TOF MS. The calculated mass of PEG is 10620 Da and
biotinylated PEG is 10902 Da.
[0054] FIG. 12A shows UPLC analysis of native Avidin.
[0055] FIG. 12B shows UPLC analysis of 1:2 mAv: Peak `a` for Avidin
is at 6.29 min, peak `b` for mAv with three PEGs is at 4.38 min,
peak `c` for mAv with two PEGs is at 5.35 min and peak `d` for mAv
with one PEG is at 5.86 min.
[0056] FIG. 13A shows the .sup.1H NMR spectrum of 8-arm PEG in
Dimethyl sulfoxide-d6. The lower-case labels "a.about.i" indicate
the resonance peaks corresponding to each proton in the
structure.
[0057] FIG. 13B shows the .sup.1H NMR spectrum of Dex in Dimethyl
sulfoxide-d6. The lower-case labels "a.about.i" indicate the
resonance peaks corresponding to each proton in the structure.
[0058] FIG. 13C shows the .sup.1H NMR spectrum of Dex-SA in
Dimethyl sulfoxide-d6. The lower-case labels "a.about.i" indicate
the resonance peaks corresponding to each proton in the
structure.
[0059] FIG. 13D shows the .sup.1H NMR spectrum of PEG-Dex-SA in
Dimethyl sulfoxide-d6. The lower-case labels "a.about.i" indicate
the resonance peaks corresponding to each proton in the
structure.
[0060] FIG. 13E shows the .sup.1H NMR spectrum of PEG-Dex-GA in
Dimethyl sulfoxide-d6. The lower-case labels "a.about.i" indicate
the resonance peaks corresponding to each proton in the
structure.
[0061] FIG. 13F shows the .sup.1H NMR spectrum of PEG-Dex-PA in
Dimethyl sulfoxide-d6. The lower-case labels "a.about.i" indicate
the resonance peaks corresponding to each proton in the
structure.
[0062] FIG. 14A relates to uptake of CPC-IOX and shows the
equilibrium uptake ratio of CPC-IOX in cartilage.
[0063] FIG. 14B relates to uptake of CPC-IOX and contains confocal
images showing depth of penetration of CPC-IOX in cartilage.
[0064] FIG. 14C relates to uptake of CPC-IOX and is a 2D color map
of CT attenuation using CPC-IOX.
[0065] FIG. 15 shows mAv-IOX produces strong CT signal in cartilage
explants with varying degree of GAG content (R squared value of
0.85) indicating that the technology can be used for staging
arthritis severity, The safranin staining in the first column shows
presence of GAG in cartilage explants.
DETAILED DESCRIPTION OF THE INVENTION
[0066] The present invention is based on the surprising discovery
that a cationic multi-arm Avidin (mAv) nano-construct enables
intra-cartilage delivery of small molecule drugs and imaging
contrast agents. The construct enables delivery of the desired
agent in high concentrations through the full thickness of the
cartilage by leveraging electrostatic interactions between the high
negative fixed charge density (FCD) of cartilage and the cationic
nano-construct.
[0067] Electrostatic interactions enable high partitioning up of
the nano-construct at the cartilage interface increasing its
concentration from C to KC, where K is the Donnan partitioning
factor (FIG. 8A-8B), which enhances its rate of penetration into
cartilage.
[0068] Typically, a contrast agent such as anionic IOX is repelled
by the negatively charged cartilage matrix resulting in
partitioning down by K'C reducing IOX's rate of transport and
uptake into the tissue. By contrast, a contrast agent conjugated to
the mAv nano-construct, with an enhanced rate of cartilage
penetration, enables high CT attenuation even at low doses for safe
diagnosis of early stage OA (FIG. 8C). The technology also has the
potential for clinical CT of other negatively charged joint soft
tissues.
[0069] Moreover, the nano-constructs of the invention enable
greater delivery of a therapeutic agent in a single low-dose of the
construct, due to the many sites on the mAv construct that may be
conjugated to the therapeutic agent. As shown herein, multi-arm
Avidin contains 28 sites for covalent conjugation of drugs (FIG. 1)
compared to 4 sites in previous designs [4]. Conjugation of
dexamethasone (Dex) as an example small molecule drug to mAv
(mAv-Dex) by using a combination of hydrolysable ester linkers
derived from succinic anhydride (SA), 3,3-dimethylglutaric
anhydride (GA) and phthalic anhydride (PA) enabled sustained
controlled release of Dex in therapeutic doses over several days.
Using in-vitro cytokine-challenged bovine cartilage models of OA,
we show that a single low dose of mAv-Dex can effectively suppress
cytokine-induced GAG loss, cell death and inflammatory response
significantly better than free (unmodified) Dex over 2 weeks. With
this multi-arm design, less than 1 .mu.M Avidin was needed--a dose
that did not cause any GAG loss or cytotoxicity. The release rate
of the conjugated therapeutic agent can be modulated by using a
combination of ester linkers with different rates of hydrolysis
based on type of drug, its target sites and the state of the
disease. This nano-construct thus has high translational potential
for enabling intra-cartilage delivery of a broad array of small
molecule OA drugs and their combinations to chondrocytes without
the previously contemplated side effects.
Definitions
[0070] The term "residue" as used herein means a portion of a
chemical structure that may be truncated or bonded to another
chemical moiety through any of its substitutable atoms. As an
example, the structure of dexamethasone is depicted below:
##STR00001##
[0071] Residues of dexamethasone include, but are not limited to,
any of the following structures:
##STR00002##
[0072] The term "hydrolyzable linking moiety" means a chemical
group that undergoes a breaking of a chemical bond when reacted
with water or a hydroxide ion under certain conditions, such as
physiological conditions.
[0073] An "active pharmaceutical ingredient" is a substance having
pharmacological activity or other effect in the treatment, cure,
diagnosis, mitigation, or prevention of a disease. The term also
encompasses substances having an effect in restoring, halting,
correcting, or modifying physiological functions.
[0074] When used in a polymeric scaffold, multi-arm polyethylene
glycol can consist of 2 repeat units of ethylene glycol up to
500,000 repeat units of ethylene glycol. In some embodiments, the
number of repeat units of PEG is from 2 to 100,000, from 2 to
50,000, from 2 to 25,000, from 2 to 10,000, from 2 to 7500, from 2
to 5000, from 2 to 2500, from 2 to 1000, from 2 to 500, from 2 to
250, from 2 to 100, from 2 to 75, or from 2 to 50 repeat units. The
average molecular weight of the PEG moiety may be about 1000 Da to
about 100,000 Da, about 5000 Da to about 50,000 Da, about 1000 Da
to about 50,000 Da, about 5000 Da to about 15,000 Da, or about
10,000 Da. The terminal groups of the multi-arm PEG used to
manufacture the scaffolds of the invention may be modified with a
functional group such as an amine. In preferred embodiments, the
PEG used in the polymeric scaffold is 8-arm PEG, more preferably
8-arm PEG-NH.sub.2.
[0075] A carbonyl group means --C(.dbd.O)--.
[0076] An amine group means --NH.sub.2 or --NH(hydrocarbyl), or
--N(hydrocarbyl).sub.2.
[0077] The term "hydrocarbyl", as used herein, refers to a group
that is bonded through a carbon atom that does not have a .dbd.O or
.dbd.S substituent, and typically has at least one carbon-hydrogen
bond and a primarily carbon backbone, but may optionally include
heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and
trifluoromethyl are considered to be hydrocarbyl for the purposes
of this application, but substituents such as acetyl (which has a
.dbd.O substituent on the linking carbon) and ethoxy (which is
linked through oxygen, not carbon) are not. Hydrocarbyl groups
include, but are not limited to aryl, heteroaryl, carbocycle,
heterocyclyl, alkyl, alkenyl, alkynyl, and combinations
thereof.
[0078] As used herein, the term "pharmaceutically acceptable" or
"pharmacologically acceptable" refers to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio. Moreover, for
animal (e.g., human) administration, it will be understood that
compositions should meet sterility, pyrogenicity, general safety
and purity standards as required by the FDA Office of Biological
Standards.
[0079] The complexes of the invention may optionally contain a
pharmaceutically acceptable excipient.
[0080] As used herein, the term "pharmaceutically acceptable
excipient" means a pharmaceutically-acceptable material,
composition or vehicle, such as a liquid or solid filler, diluent,
excipient, manufacturing aid (e.g., lubricant, talc magnesium,
calcium or zinc stearate, or steric acid), or solvent encapsulating
material, involved in carrying or transporting the subject compound
from one organ, or portion of the body, to another organ, or
portion of the body. Each carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not injurious to the patient. Some examples of materials which
can serve as pharmaceutically-acceptable excipients include: (1)
sugars, such as lactose, glucose and sucrose; (2) starches, such as
corn starch and potato starch; (3) cellulose, and its derivatives,
such as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents,
such as magnesium stearate, sodium lauryl sulfate and talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl
oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; (22) bulking agents, such as
polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; (22) C.sub.2-C.sub.12 alcohols, such as
ethanol; and (23) other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, coloring agents,
release agents, coating agents, disintegrating agents, binders,
sweetening agents, flavoring agents, perfuming agents, protease
inhibitors, plasticizers, emulsifiers, stabilizing agents,
viscosity increasing agents, film forming agents, solubilizing
agents, surfactants, preservative and antioxidants can also be
present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable excipient" or the like are
used interchangeably herein.
[0081] The present invention also contemplates pharmaceutically
acceptable salts of the compounds of the invention. In certain
embodiments, contemplated salts of the invention include, but are
not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium
salts. In certain embodiments, contemplated salts of the invention
include, but are not limited to, L-arginine, benenthamine,
benzathine, betaine, calcium hydroxide, choline, deanol,
diethanolamine, diethylamine, 2-(diethylamino)ethanol,
ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine,
1H-imidazole, lithium, L-lysine, magnesium,
4-(2-hydroxyethyl)morpholine, piperazine, potassium,
1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,
tromethamine, and zinc salts. In certain embodiments, contemplated
salts of the invention include, but are not limited to, Na, Ca, K,
Mg, Zn or other metal salts.
[0082] The pharmaceutically acceptable acid addition salts can also
exist as various solvates, such as with water, methanol, ethanol,
dimethylformamide, and the like. Mixtures of such solvates can also
be prepared. The source of such solvate can be from the solvent of
crystallization, inherent in the solvent of preparation or
crystallization, or adventitious to such solvent.
[0083] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
Preferably the administration is intraarterial.
[0084] Pharmaceutical compositions suitable for parenteral
administration comprise one or more active compounds in combination
with one or more pharmaceutically acceptable sterile isotonic
aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0085] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0086] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0087] For use in the methods of this invention, active compounds
can be given per se or as a pharmaceutical composition containing,
for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0088] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinacious
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of a compound at a particular target site.
[0089] Actual dosage levels of the active ingredients in the
pharmaceutical compositions may be varied so as to obtain an amount
of the active ingredient that is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient.
[0090] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound or
combination of compounds employed, or the ester, salt or amide
thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound(s) being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compound(s)
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0091] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the therapeutically effective
amount of the pharmaceutical composition required. For example, the
physician or veterinarian could start doses of the pharmaceutical
composition or compound at levels lower than that required in order
to achieve the desired therapeutic effect and gradually increase
the dosage until the desired effect is achieved. By
"therapeutically effective amount" is meant the concentration of a
compound that is sufficient to elicit the desired therapeutic
effect. It is generally understood that the effective amount of the
compound will vary according to the weight, sex, age, and medical
history of the subject. Other factors which influence the effective
amount may include, but are not limited to, the severity of the
patient's condition, the disorder being treated, the stability of
the compound, and, if desired, another type of therapeutic agent
being administered with the compound of the invention. A larger
total dose can be delivered by multiple administrations of the
agent. Methods to determine efficacy and dosage are known to those
skilled in the art (Isselbacher et al. (1996) Harrison's Principles
of Internal Medicine 13 ed., 1814-1882, herein incorporated by
reference).
[0092] In general, a suitable daily dose of an active compound used
in the compositions and methods of the invention will be that
amount of the compound that is the lowest dose effective to produce
a therapeutic effect. Such an effective dose will generally depend
upon the factors described above.
[0093] If desired, the effective daily dose of the active compound
may be administered as one, two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. In certain
embodiments of the present invention, the active compound may be
administered two or three times daily. In preferred embodiments,
the active compound will be administered once daily.
[0094] The patient receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in
general.
Embodiments of the Invention
[0095] The present invention provides a platform for the delivery
of small molecule drugs or contrast agents to joints and other soft
tissues. The platform enables penetration of the drug through the
full thickness of cartilage and long intra-cartilage residence time
by leveraging electrostatic interactions between the cationic
platform and the anionic cartilage matrix. Embodiments of the
invention are described below.
[0096] In certain embodiments, the present invention provides a
compound, comprising: [0097] a residue of biotin; [0098] a
multi-arm polymeric scaffold; [0099] one or more hydrolyzable
linking moieties; and [0100] one or more residues of an active
pharmaceutical ingredient; [0101] wherein the residue of biotin is
covalently attached to the multi-arm polymeric scaffold; [0102]
each of the one or more hydrolyzable linking moieties are
covalently attached to the multi-arm polymeric scaffold; and [0103]
each of the one or more residues of the active pharmaceutical
ingredient are covalently attached to each of the one or more
hydrolyzable linking moieties.
[0104] In certain embodiments, the residue of biotin is covalently
attached to the multi-arm polymeric scaffold on a different arm
than the one or more hydrolyzable linking moieties.
[0105] In certain embodiments, the compound comprises two or more
hydrolyzable linking moieties, wherein each hydrolyzable linking
moiety is covalently attached to a different arm of the multi-arm
polymeric scaffold.
[0106] In certain embodiments, one arm of the multi-arm polymeric
scaffold is covalently attached to the residue of biotin, and each
of the remaining arms of the multi-arm polymeric scaffold is
attached to a hydrolyzable linking moiety, and each hydrolyzable
linking moiety is attached to a residue of an active pharmaceutical
ingredient.
[0107] In certain embodiments, the multi-arm polymeric scaffold
comprises at least 3 arms. In certain embodiments, multi-arm
polymeric scaffold comprises at least 4 arms or at least 6 arms. In
certain embodiments, the multi-arm polymeric scaffold comprises 8
arms. In certain such embodiments, the compound comprises 8
hydrolyzable linking moieties and 8 residues of an active
pharmaceutical ingredient.
[0108] In certain embodiments, the multi-arm polymeric scaffold
comprises a hydrophilic polymer. Exemplary hydrophilic polymers
useful in the compounds of the invention include polyacrylamide,
polyurethanes, poly (hydroxyethyl methacrylamide), and polyethylene
glycol. In certain embodiments, the multi-arm polymeric scaffold
may be a multi-arm polyethylene glycol scaffold.
[0109] Multi-arm polymers encompass numerous configurations that
are useful in the compounds, complexes, and methods of the
invention. These configurations include star polymers,
hyperbranched polymers, dendrimers, graft polymers, and polymer
networks.
[0110] In certain embodiments, the polymeric scaffold comprises a
star polymers. Star polymers include, but are not limited to,
copolymers based on polyethylene oxide (PEO) and poly(methyl
methacrylate) (PMMA); copolymers based on PMMA-PAA (poly(acrylic
acid)); N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers;
poly(styrene) core and poly(tert-butyl acrylate) arms; thiol
terminated multi-arm (6 and 8) poly(ethylene-glycol) (PEG); also
PEG-PAMAM. In the complexes of the invention (e.g., in which avidin
is the core), such polymers form core cross-linked star
polymers.
[0111] In certain embodiments, the polymeric scaffold comprises a
hyperbranched polymer. Hyperbranched polymers may include monomers
such as polyester, polyesteramine, polyamine, polyester,
polyglycerols, polyphosphate, polyglycerols-PEG.
[0112] In certain embodiments, the polymeric scaffold comprises a
polypeptide system including polycationic conjugates poly [Lys
(DL-Alam-Leui)] (ALK), poly [Lys(Ser-DL-Ala3)] (SAK) amphoteric
poly[Lys(Glu-DL-Ala3)] (EAK). In further embodiments, the polymeric
scaffold comprises a polymer such as Poly (beta-amino ester) (PBAE)
or Chitosan.
[0113] In certain embodiments, each of the one of more hydrolyzable
linking moieties comprises a carbonyl group. In further
embodiments, each of the one of more hydrolyzable linking moieties
is selected from the group consisting of
##STR00003##
In certain embodiments, each of the one of more hydrolyzable
linking moieties is
##STR00004##
Alternatively, each of the one of more hydrolyzable linking
moieties may be
##STR00005##
Alternatively, each of the one of more hydrolyzable linking
moieties is
##STR00006##
[0114] In certain embodiments, the active pharmaceutical ingredient
is a glucocorticoid or pharmaceutically acceptable salt thereof.
Exemplary glucocorticoids include cortisol, cortisone, prednisone,
prednisolone, methylprednisolone, dexamethasone, betamethasone,
triamcinolone, fludrocortisone, deoxycorticosterone, aldosterone,
and beclometasone. In certain embodiments, the glucocorticoid is
dexamethasone, triamcinolone, or prednisone; preferably
dexamethasone.
[0115] In further embodiments, the active pharmaceutical agent is a
protein, such as insulin link growth factor-1 (IGF-1).
[0116] In certain embodiments, the multi-arm polymeric scaffold
comprises an 8-arm polyethylene glycol scaffold;
each of the one of more hydrolyzable linking moieties is selected
from the group consisting of
##STR00007##
and
[0117] the active pharmaceutical ingredient is dexamethasone.
[0118] In certain embodiments, the compound has the following
structure:
##STR00008##
wherein X is a hydrolyzable linking moiety selected from the group
consisting of
##STR00009##
and n is an integer from 1 to 500,000; or a pharmaceutically
acceptable salt thereof.
[0119] In further embodiments, the compound has the following
structure:
##STR00010##
wherein X is a hydrolyzable linking moiety selected from the group
consisting of
##STR00011##
and n is an integer from 1 to 500,000; or a pharmaceutically
acceptable salt thereof.
[0120] In further aspects, the present invention provides a complex
comprising avidin and one or more compounds described herein. In
certain embodiments, the one or more compounds are bound to avidin
through electrostatic interactions.
[0121] In certain embodiments, avidin and the one or more compounds
are in a molar ratio of 1:4; and all four biotin-binding sites of
avidin are bound to one of the one or more compounds.
[0122] In certain embodiments, the complex comprises four compounds
described herein, wherein the four compounds are identical to each
other.
[0123] In certain embodiments, the complex comprises four compounds
described herein, wherein at least one compound is different from
the other compounds.
[0124] In certain embodiments, the complex comprises four compounds
described herein, wherein at least two compounds are different from
each other and from the other compounds.
[0125] In certain embodiments, the complex comprises four compounds
described herein, wherein all four compounds are different from
each other.
[0126] In certain embodiments of the complex comprising four
compounds of the invention, in all four compounds X is
##STR00012##
[0127] In certain embodiments of the complex comprising four
compounds of the invention, in all four compounds X is
##STR00013##
[0128] In certain embodiments of the complex comprising four
compounds of the invention, in all four compounds X is
##STR00014##
[0129] In certain embodiments of the complex comprising four
compounds of the invention, in two compounds X is in one compound X
is
##STR00015##
and in one
##STR00016##
compound X is
##STR00017##
[0130] The present invention also provides methods of treating a
joint disease in a subject in need thereof, comprising
administering to the subject a compound described herein or a
complex described herein. In certain embodiments, the method
comprises administering a complex described herein.
[0131] In certain embodiments, the joint disease is selected from
the group consisting of rheumatoid arthritis, spondyloarthritis,
juvenile idiopathic arthritis, lupus, gout, bursitis, and
osteoarthritis. In certain embodiments, the joint disease is
osteoarthritis.
[0132] In certain embodiments, administering a compound or complex
described herein comprises intra-articular injection.
[0133] The present invention also provides methods of preventing
glycosaminoglycan (GAG) loss in a subject in need thereof,
comprising administering to the subject a compound described herein
or a complex described herein. In certain embodiments, the method
comprises administering a complex described herein.
[0134] The present invention also provides compounds, comprising:
[0135] a residue of biotin; [0136] a multi-arm polymeric scaffold;
and one or more residues of a contrast agent; [0137] wherein the
residue of biotin is covalently attached to the multi-arm polymeric
scaffold; and [0138] each of the one or more residues of a contrast
agent are covalently attached to the multi-arm polymeric
scaffold.
[0139] In certain embodiments, the residue of biotin is covalently
attached to the multi-arm polymeric scaffold on a different arm
than the one or more residues of a contrast agent.
[0140] In certain embodiments, the compound comprises two or more
residues of a contrast agent, wherein each residue of a contrast
agent is covalently attached to a different arm of the multi-arm
polymeric scaffold.
[0141] In certain embodiments, one arm of the multi-arm polymeric
scaffold is covalently attached to the residue of biotin, and each
of the remaining arms of the multi-arm polymeric scaffold is
attached to a residue of a contrast agent.
[0142] In certain embodiments, the multi-arm polymeric scaffold
comprises at least 3 arms. In certain embodiments, multi-arm
polymeric scaffold comprises at least 4 arms or at least 6 arms. In
certain embodiments, the multi-arm polymeric scaffold comprises 8
arms. In certain such embodiments, the compound comprises 8
residues of a contrast agent.
[0143] In certain embodiments, the multi-arm polymeric scaffold
comprises a hydrophilic polymer. Exemplary hydrophilic polymers
useful in the compounds of the invention include polyacrylamide,
polyurethanes, poly (hydroxyethyl methacrylamide), and polyethylene
glycol.
[0144] In certain embodiments, the multi-arm polymeric scaffold may
be a multi-arm polyethylene glycol scaffold.
[0145] In certain embodiments, the contrast agent is a radiopaque
contrast agent or pharmaceutically acceptable salt thereof.
Exemplary contrast agents include diatrizoate, metrizoate,
iothalamate, and ioxaglate. Preferably the contrast agent is
ioxaglate.
[0146] In certain embodiments, the compound has the structure:
##STR00018##
n is an integer from 1 to 500,000; or a pharmaceutically acceptable
salt thereof.
[0147] In further aspects, the present invention provides a complex
comprising avidin and one or more compounds described herein. In
certain embodiments, the one or more compounds are bound to avidin
through electrostatic interactions.
[0148] In certain embodiments, avidin and the one or more compounds
are in a molar ratio of 1:4; and all four biotin-binding sites of
avidin are bound to one of the one or more compounds.
[0149] In certain embodiments, the complex comprises four compounds
described herein, wherein the four compounds are identical to each
other.
[0150] In certain embodiments, the complex comprises four compounds
described herein, wherein at least one compound is different from
the other compounds.
[0151] In certain embodiments, the complex comprises four compounds
described herein, wherein at least two compounds are different from
each other and from the other compounds.
[0152] In certain embodiments, the complex comprises four compounds
described herein, wherein all four compounds are different from
each other.
[0153] The present invention further provides a method of
diagnosing a joint disease, comprising administering to a subject a
compound or a complex described herein; imaging the joint, and
assessing the resultant images to determine whether the subject is
suffering from a joint disease.
[0154] Exemplary joint diseases include rheumatoid arthritis,
spondyloarthritis, juvenile idiopathic arthritis, lupus, gout,
bursitis, and osteoarthritis. Preferably the method is a method for
diagnosing osteoarthritis.
[0155] In certain embodiments, administering a compound or complex
described herein comprises intra-articular injection.
[0156] In certain embodiments, the step of imaging comprises
computed tomography (CT) imaging.
[0157] The method also provides a method for imaging soft tissue,
comprising administering to a subject a compound or a complex
described herein, and imaging the soft tissue. In certain
embodiments, the soft tissue is cartilage. In certain embodiments,
the step of imaging comprises computed tomography (CT) imaging.
[0158] The present invention also provides a complex, comprising:
[0159] a cationic protein; and [0160] one or more residues of a
contrast agent; [0161] wherein the residue of the contrast agent is
covalently attached to protein.
[0162] In certain embodiments, the cationic protein is avidin.
[0163] In certain embodiments, the contrast agent is a radiopaque
contrast agent or pharmaceutically acceptable salt thereof.
Exemplary contrast agents include diatrizoate, metrizoate,
iothalamate, and ioxaglate. Preferably the contrast agent is
ioxaglate.
[0164] The present invention further provides a method of
diagnosing a joint disease, comprising administering to a subject a
compound or a complex described herein; imaging the joint, and
assessing the resultant images to determine whether the subject is
suffering from a joint disease.
[0165] Exemplary joint diseases include rheumatoid arthritis,
spondyloarthritis, juvenile idiopathic arthritis, lupus, gout,
bursitis, and osteoarthritis. Preferably the method is a method for
diagnosing osteoarthritis.
[0166] In certain embodiments, administering a compound or complex
described herein comprises intra-articular injection.
[0167] In certain embodiments, the step of imaging comprises
computed tomography (CT) imaging.
[0168] The method also provides a method for imaging soft tissue,
comprising administering to a subject a compound or a complex
described herein, and imaging the soft tissue. In certain
embodiments, the soft tissue is cartilage. In certain embodiments,
the step of imaging comprises computed tomography (CT) imaging.
Cationic Peptide Carriers for Cartilage Targeting
[0169] Targeted drug delivery to negatively charged tissues in the
body such as cartilage remains an outstanding challenge. The high
negative fixed charged density (FCD) of these tissues prevents
effective diffusion of therapeutics to reach their target sites
within the tissue. However, this high negative FCD can be utilized
to enhance transport of therapeutics in such tissues through
electrostatic interactions by making them positively charged. The
concentration of positively charged carriers at the interface of
negatively charged tissues is enhanced by a factor of K.sup.+,
known as Donnan partitioning coefficient compared to the
surrounding bath of cationic solute (K.sup.+C vs C). This sharp
increase in concentration of the cationic carrier at the tissue
interface (K.sup.+C) creates a higher concentration gradient along
the tissue thickness which results in faster diffusion rates across
the tissue. It is crucial to engineer the net positive charge of
the carrier based on the negative FCD of the tissue to modulate the
binding strength of the cationic carrier with the negatively
charged tissue matrix to allow full thickness penetration using
weak and reversible charge interactions. Too high of a positive
charge will result in too strong binding that will hamper diffusion
of the carrier into the tissue. Therefore, modulating the net
positive charge of the carrier is essential to ensure full
thickness penetration using weak reversible charge interactions,
while the binding is strong enough to allow for high uptake and
retention of the carrier in the tissue.
[0170] The present invention provides a charge based approach to
modify therapeutic agents or contrast agents with optimally charged
cationic peptide carriers (CPCs) for targeted delivery to a tissue
of known fixed charge density (FCD). The invention is based in part
on the surprising discovery of cartilage penetrating CPCs of
varying net charge, spatial distribution and hydrophobicity (see
table below) that enable delivery of a variety of macromolecules
(including therapeutic drugs and contrast agents) inside cartilage.
Studies presented herein and in Acta Biomater. (2018)
doi:10.1016/j.actbio.2018.12.004 show that there exists an optimal
charge range for a solute of given size to effectively target a
tissue of known FCD.
TABLE-US-00001 Cationic peptide carrier (CPC) sequences and net
charge Net Cationic Peptide SEQ charge (z) Carrier Sequence ID NO:
+8 RRAAAARRAAAARRAAAARR 1 +14 RRRRAARRRAARRRAARRRR 2 +16
(ARRRAARA).sub.4 3 +20 RRRRRRRRRRRRRRRRRRRR 4
[0171] As detailed in Acta Biomater. (2018)
doi:10.1016/j.actbio.2018.12.004, CPC uptake increased with
increasing net charge from +8 to +14. However, as charge increased
further, the intra-cartilage penetration decreased likely due to
stronger binding interactions that hindered CPC penetrability and
uptake. This confirms that that weak-reversible binding is
essential to enable penetration of CPCs through full tissue
thickness.
[0172] In certain embodiments, CPC +8 is the carrier used for
cartilage imaging due to its very fast transport rate, full depth
penetration and shorter intra-tis sue residence time, which can
help clear contrast agents from the joint post imaging. The optimal
charge of CPC +8 allows rapid transport in cartilage with short
residence time which is ideal for delivery of imaging contrast
agents. Additionally, upon incubation of CPC +8 with chondrocytes
in culture, CPC +8 is not uptaken by the chondrocytes, which has
clinical importance as for cartilage imaging purposes, presence of
the contrast agents needs to be limited to the cartilage matrix
rather than inside the cells.
[0173] CPC +14 had the highest intra-cartilage uptake among the
CPCs, demonstrated high intra-cartilage retention while maintain
full-thickness penetration. The optimal charge of CPC +14 allowed
for stronger binding with cartilage GAGs without significantly
reducing its transport rate. Consequently, the high uptake, high
retention and full thickness diffusion of CPC +14 make it an ideal
carrier for drug delivery applications. Thus, in certain
embodiments, CPC +14 is the carrier used for delivering therapeutic
agents to cartilage.
[0174] CPCs are shown to be safe for cell and cartilage matrix
health with no harmful effects on rate of GAG loss, cell viability
and rate of GAG synthesis by chondrocytes even at high
concentrations (Acta Biomater. (2018)
doi:10.1016/j.actbio.2018.12.004).
[0175] The present invention provides a method of delivering a
contrast agent or an active pharmaceutical ingredient to a
negatively charged tissue in a subject, comprising administering to
the subject a complex;
wherein the complex comprises a residue of a contrast agent or an
active pharmaceutical ingredient and a cationic peptide, wherein
the peptide comprises from 2 to 40 amino acid residues, and the net
charge of the peptide is from +7 to +20 inclusive; and the residue
of the contrast agent or the active pharmaceutical ingredient is
covalently bonded to the peptide.
[0176] In certain embodiments, the peptide comprises at least one
arginine residue, lysine residue, or other positively-charged amino
acid residue. In certain embodiments, the peptide comprises at
least one arginine residue or lysine residue.
[0177] Exemplary peptides include:
TABLE-US-00002 (SEQ ID NO: 1) RRAAAARRAAAARRAAAARR; (SEQ ID NO: 2)
RRRRAARRRAARRRAARRRR; (SEQ ID NO: 3) (ARRRAARA).sub.4; and (SEQ ID
NO: 4) RRRRRRRRRRRRRRRR RRRR.
[0178] In certain embodiments, the residue of the contrast agent or
the active pharmaceutical ingredient is covalently bonded to the
peptide at an arginine residue.
[0179] In certain embodiments, the peptide is RRAAAARRAAAARRAAAARR
(SEQ ID NO: 1). In certain such embodiments, the complex comprises
a residue of a contrast agent. Exemplary contrast agents include
diatrizoate, metrizoate, iothalamate, and ioxaglate. In certain
embodiments, the contrast agent is ioxaglate.
[0180] In certain embodiments, the peptide is RRRRAARRRAARRRAARRRR
(SEQ ID NO: 2). In certain such embodiments, the complex comprises
a residue of an active pharmaceutical ingredient. In certain
embodiments, the active pharmaceutical ingredient is a protein such
as IGF-1.
Examples
[0181] Materials
[0182] 10 kDa 8-arm polyethylene glycol (PEG) amine hydrochloride
salt was purchased from Advanced Biochemicals (Lawrenceville, Ga.).
N-Hydroxysuccinimido (NHS)-biotin, 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide hydrochloride (EDC),
N-hydroxysulfosuccinimide (NHSS), Avidin and Avidin-Texas Red
conjugated, 4'-hydroxybenzene-2-carboxylic acid (HABA), 3.5 kDa
molecular weight cut-off (MWCO), 7.0 kDa MWCO SnakeSkin dialysis
tubing was purchased from Thermo Fisher Scientific (Waltham,
Mass.). Proteinase-K was purchased from Roche Diagnostics
(Risch-Rotkreuz, Switzerland). Dulbecco's Modification of Eagle's
Medium (DMEM) was from Cellgro (Manassas, Va.). HEPES,
non-essential amino acids (NEAA), penicillin-streptomycin
Antibiotic-Antimycotic (PSA) and trypsin-EDTA phenol red were
purchased from Gibco (Carlsbad, Calif.). Ascorbic acid and
L-proline were from Fisher Bioreagents (Pittsburgh, Pa.). Propidium
iodide (PI) was obtained from Thermofisher Acros Organics (Geel,
Belgium). Human recombinant IL-1.alpha. was from PeproTech (Rocky
Hill, N.J.). Antibodies for type II collagen immunohistochemistry
was acquired from the Developmental Studies Hybridoma Bank
(University of Iowa), while the Vectastain Elite ABC kit was from
Vector Laboratories (Burlingame, Calif.). Dex, SA, GA, PA, dimethyl
sulfoxide-d6 (DMSO-d6) containing 0.03% (v/v) tetramethylsilane,
fluorescein diacetate (FDA), fluorescein isothiocyanate isomer I
(FITC), dimethylaminopyridine (DMAP), resazurin sodium salt, Griess
reagent, histology reagents and other salts were purchased from
Sigma-Aldrich (St. Louis, Mo.).
Chemical Synthesis
A. Biotinylation of 8-Arm PEG
[0183] 10 kDa PEG was biotinylated by reaction with NHS-biotin.
Briefly, 10 mg (0.001 mmol, 1.0 equiv.) of PEG was dissolved in 500
.mu.L of nanopure water and 1.7 mg (0.005 mmol, 5.0 equiv.) of
NHS-biotin was dissolved in 500 .mu.L of DMSO. NHS-biotin solution
was then added dropwise to the PEG solution (5:1 molar ratio) and
reacted for 2 h under gentle rotation at room temperature using
click chemistry between the NHS group in biotin and amine groups in
PEG. Excess NHS-biotin was removed from the PEG-biotin conjugate
solution using dialysis (7.0 kDa MWCO) for 24 h against phosphate
buffer saline (PBS). Extent of biotinylation was confirmed using
the HABA dye assay [5, incorporated by reference].
B. Synthesis of Dexamethasone Hemisuccinate (Dex-SA), Glutarate
(Dex-GA) and Phthalate (Dex-PA)
[0184] Three carboxylated derivatives of Dex were prepared by
reacting 36.0 mg Dex (0.092 mmol, 1.0 equiv.) with 46 mg of SA,
52.0 mg of GA or 67.0 mg of PA (0.458 mmol, 5.0 equiv.) in presence
of 2 mg DMAP (0.015 mmol, 0.2 equiv.) as a catalyzer in 1 mL of
pyridine. The reaction for Dex-SA was conducted in a round bottom
flask purged with nitrogen gas for 24 h at room temperature. For
Dex-GA and Dex-PA, the reaction time was 48 h at 37.degree. C.
Following completion of the reaction, pyridine was evaporated with
constant purging of nitrogen gas, and 4 mL of the cold solution
containing 25 mL water and 10 mL concentrated hydrochloride acid
(HCl) was added to the flask to precipitate Dex-SA, Dex-GA and
Dex-PA out. A white precipitate was observed, which was stirred for
10 min and then centrifuged at 10,000 g for 5 min for 5 cycles. In
each cycle, the supernatant was replaced with fresh cold solution
of HCl. The final products of Dex-SA, Dex-GA, and Dex-PA were
lyophilized, weighed and stored at -20.degree. C. for future use.
Their structures were confirmed using Proton Nuclear Magnetic
Resonance (.sup.1H-NMR). The carboxyl groups incorporated to the
Dex were verified with thin layer chromatography (TLC).
C. Conjugation of Dex-SA, Dex-GA and Dex-PA to PEG-Biotin
[0185] Dex-SA was conjugated to PEG-biotin using EDC/NHS chemistry.
Briefly, 5.0 mg of Dex-SA, Dex-GA or Dex-PA (0.010 mmol, 100.0
equiv.) was dissolved initially in 120 .mu.L of DMSO and added 600
.mu.L of 2-morpholinoethanesulfonic acid (MES) dropwise. Then, 19.2
mg of EDC (0.104 mmol, 104.0 equiv.) and 21.7 mg NHSS (0.092 mmol,
92.0 equiv.) were added to Dex-SA, Dex-GA and Dex-PA solution, and
all of them were purged with nitrogen to activate the reaction for
30 min. Subsequently, 1.0 mg of PEG-biotin (0.100 .mu.mol, 1.0
equiv.) was added to each of the solutions and reacted for 2 h at
room temperature, purged with nitrogen gas. Upon completion of the
reaction, the final product was dialyzed using 7.0 kDa MWCO
membrane to remove the excessive reagents under 4.degree. C. for 24
h. The pure product was then lyophilized and stored at -20.degree.
C. for future purposes. The formation of these three chemical
compounds was then confirmed using .sup.1H-NMR.
D. Conjugation of Ioxaglate (IOX) to PEG-Biotin
##STR00019## ##STR00020##
[0187] D1: Synthesis of Intermediate Product, Ioxaglate Chloride
(IOX-Cl) [0188] 1. Weigh 12.7 mg of IOX (0.01 mmol, 20.0 equiv.) in
1.5 mL Eppendorf tube and suspend IOX with 110 .mu.L thionyl
chloride (1.5 mmol, 3000.0 equiv.) to form a gray insoluble
mixture. [0189] 2. Add 5 .mu.L DMF to mixture as a catalyst. [0190]
3. Purge nitrogen gas to remove any moisture inside the tube and
use parafilm to seal tube cap. [0191] 4. Keep mixture reacting for
6 h at 70.degree. C. using Thermal Shaker, with 600 rpm shaking
speed. IOX will dissolve at high temperature producing a brown
solution. [0192] 5. After the reaction, cool down the synthesized
intermediate product (IOX-Cl). To remove excessive unreacted
thionyl chloride, keep the product under vacuum at 20.degree. C.
Intermittent slight heating of thionyl chloride to 40.degree. C.
will also facilitate its evaporation. To protect the vacuum pump,
thionyl chloride vapor should pass through a gas washing bottle
containing 1 N NaOH solution. [0193] 6. Add 200 .mu.L DMSO to
dissolve the remaining solid and transfer the solution to a new 1.5
mL Eppendorf tube for lyophilization. [0194] 7. Dissolve 5 mg of
standard IOX and IOX-Cl in 700 .mu.L DMSO-d6 for 1H-NMR,
respectively. The structure of IOX-Cl can be verified using 500 MHz
.sup.1H-NMR. The disappearance of hydroxyl peak and carboxyl peak
in IOX-Cl compared to the IOX standard, confirms that hydroxyl and
carboxyl groups of IOX have been chloritized.
D2: Conjugation of IOX-Cl to 8-Arm Biotin-PEG to Synthesize
Biotin-PEG-IOX
[0194] [0195] 1. Synthesize biotinylated 8-arm PEGs as outlined in
section A, above. [0196] 2. To conjugate IOX-Cl to 8-arm
biotin-PEG, first dissolve 13.1 mg of the lyophilized product,
IOX-Cl (0.01 mmol, 20 equiv.) in 300 .mu.L of anhydrous DMF using
1.5 mL Eppendorf tube. [0197] 3. Add 7.5 .mu.L TEA dropwise to the
solution from step 2. Keep this solution at 4.degree. C. using a
Thermal Shaker. [0198] 4. Dissolve 5.5 mg of 8-arm biotin-PEG in
100 .mu.L DMF (0.0005 mmol, 1 equiv.) to make 8-arm biotin-PEG
solution. Then, add it dropwise to the IOX-Cl solution from step 3.
[0199] 5. Conduct this reaction in Thermal shaker at 4.degree. C.,
600 rpm for 30 min and then at 50.degree. C., 600 rpm for 16 h.
[0200] 6. After the reaction, transfer the reacted product (8-arm
biotin-PEG-IOX) to 4 mL ice-water in order to precipitate unreacted
IOX-Cl. Centrifuge the mixture at 10,000 g for 5 min and collect
the supernatant. [0201] 7. To further purify the product, add
collected supernatant to 3.5 kDa dialysis membrane and then dialyze
against PBS for 24 h and DI water for another 24 h at room
temperature. [0202] 8. Then lyophilize the purified product, 8-arm
biotin-PEG-IOX and freeze at -80.degree. C. for future
applications. [0203] 9. To estimate the amount of IOX loaded on
8-arm biotin-PEG, use HPLC equipped with a Variable Wavelength
Detector and an Advance Bio RP-mAb C4 4.6.times.150 mm column.
Prepare a gradient of solvent A (0.1% TFA in water) and solvent B
(0.1% TFA in acetonitrile). Linearly increase the concentration of
solvent B from 5% to 65% over 26 min. Set column temperature at
45.degree. C. and a flow rate of 1.0 mL/min. Standard IOX will
elute at 3.710 min, and 8-arm biotin-PEG-IOX will be eluted between
18 to 24 min (254 nm) as PEG polymer does not have constant
repeating units and the amount of IOX is not conjugated evenly to
PEG. Estimate the total UV absorbance (mAU*s) of PEG-IOX by
integrating the area under the curve and then estimate IOX
concentration according to IOX standard curve. About an average of
3.82.+-.0.50 moles of IOX conjugated to one mole of 8-arm
biotin-PEG is expected. [0204] 10. Use MALDI-TOF to confirm the
increase in molecular weight of 8-arm biotin-PEG-IOX compared to
8-arm biotin-PEG. Each mole of 8-arm biotin-PEG is conjugated to
2.33 moles of IOX on an average. E. Loading of Dex-PEG-Biotin on
Avidin to Synthesize mAv-Dex
[0205] The three Dex-PEG-biotin products from section C, above,
were reacted with Avidin in nanopure water for 30 min under gentle
shaking at room temperature in 1:1, 2:1, 3:1, 4:1, 5:1, 6:1 and 8:1
molar ratios to determine the ratio at which all four biotin
binding sites of Avidin are occupied by Dex-PEG-biotin to form 1:4
mAv, which was confirmed using the HABA dye assay. For subsequent
drug release and biological studies, we have used 1:4 mAv
configuration as it has higher number of sites for drug loading
than 1:2 mAv and it has been referenced simply as mAv in following
text. In one formulation, mAv-Dex-SA, mAv-Dex-GA, and mAv-Dex-PA
were physically mixed together in 2:1:1 molar ratio of Dex,
abbreviated as controlled release mAv-Dex (2:1:1). We compared its
bioactivity with mAv-Dex-SA, which is referred as fast release
mAv-Dex throughout the manuscript. The products were then
lyophilized and stored at -20.degree. C. until further use.
F. Loading of Biotin-PEG-IOX on Avidin to Synthesize mAv-IOX
[0206] Biotin-PEG-IOX from Section D may be reacted with Avidin
according to the method outlined in Section E.
Application in Computed Tomography of Cartilage
[0207] 1. Use the procedure in Section F to conjugate 4 moles of
biotin-PEG-IOX with Avidin at its biotin binding sites to
synthesize mAv-IOX. [0208] 2. Use Zetasizer Nano-ZS90 to detect the
zeta potential of unmodified Avidin and mAv-IOX in DI water; the
zeta potential (.xi., mV) of Avidin and mAv-IOX should be around
18.3.+-.0.5 mV and 8.69.+-.0.7 mV, respectively. [0209] 3. Obtain 3
mm diameter.times.1 mm height cylindrical cartilage discs with
intact superficial zone (SZ) from femoropatellar grooves of
1-2-week-old bovine knee joints and freeze them in PBS containing
protease inhibitors (PI) until further use. [0210] 4. Prepare (A)
0.5 mg I/mL IOX solution; (B) 16 mg I/mL IOX solution; (C) 0.5 mg
I/mL mAv-IOX solution using PBS/PI (dissolve 1 tablet PI in 50 mL
PBS buffer) as solvent. Use a freezing point osmometer to balance
the osmolality of these contrast agent solutions to 400.+-.20
mOsm/kg by adding 1 M sodium chloride solution dropwise. [0211] 5.
Incubate 3 mm diameter cartilage explants in 96-well plate with 300
.mu.L of different iodinated contrast agent solutions (0.5 mg I/mL
IOX; 16 mg I/mL IOX and 0.5 mg I/mL mAv-IOX) for 24 h at 37.degree.
C. using an incubator. [0212] 6. Secure cartilage explants using
low X-ray-absorption foam inside a 20.5 mm CT scan tube. Ensure
that the cartilage explant stays secured in the foam during CT
imaging. [0213] 7. Take one of the equilibrated cartilage explants
from contrast agent solution and gently using Kimwipes to remove
solution from the tissue surface. [0214] 8. Set .mu.CT35 with an
isotropic voxel resolution of 10 .mu.m, 70 kVp tube voltage, 113
.mu.A current, and 200 ms integration time method. The CT data will
be saved as DICOM format. Image) software will automatically
display Hounsfield Unit (HU) by importing DICOM images. [0215] 9.
Process the CT imaging data acquired from .mu.CT35 using ImageJ
software: (i) Go to Image.fwdarw.Stacks.fwdarw.Images to Stack to
convert CT imaging data to a cartilage stack. (ii) Go to
Image.fwdarw.Stacks.fwdarw.Reslice and select `starting at top` to
acquire a new stack showing vertical section of cartilage from
superficial zone (SZ) to deep zone (DZ). The output spacing (mm) is
0.010 and select `Avoid interpolation`. (iii) Go to
Image.fwdarw.Adjust.fwdarw.Threshold and apply range as -300 to
+.infin.. Any intensities below -300, which is mostly background,
will be set to `NaN`. (iv) Use an ImageJ plugin (use the link in
Note 8) to acquire Z-projection of average intensity of the
cartilage stack. The cartilage stack will be converted to one image
with average intensity. (v) Go to LUT416 colors to create a color
map of cartilage. (vi) Go to
Image.fwdarw.Adjust.fwdarw.Brightness/Contrast and set 0 minimum
and 700 Maximum to show higher contrast among different intensities
in cartilage color map. (vii) Go to
Analyze.fwdarw.Tools.fwdarw.Calibration Bar to add a calibration
color bar. [0216] 10. The color maps of contrast agent spatial
distributions in cartilage explants from SZ to DZ are shown in FIG.
9. 0.5 mg I/mL mAv-IOX produced significantly high CECT attenuation
with intensity increasing with GAG density from SZ to DZ of
cartilage. Similar IOX concentration did not show any signal and
32.times. high concentration of IOX (16 mg I/mL) was needed to
produce similar CT attenuation.
Estimating Correlation Between Contrast Enhanced CT Attenuation
(CECT) and GAG Density in Cartilage
[0216] [0217] 1. Incubate 3 mm diameter cartilage explants in
96-well plates with saline as blank control, 0.1 U/mL
chondroitinase-ABC for 8 h and 16 h, 0.25 U/mL chondroitinase-ABC
for 16 h and 24 h in order to induce varying degrees of GAG
depleted cartilage to simulate different OA stages (early, mid and
late stage). After digestion of chondroitinase-ABC, wash each
cartilage with PBS/PI for 3 times. [0218] 2. Incubate these
explants in 0.5 mg I/mL mAv-IOX and 16 mg I/mL IOX to enhance CT
imaging of these cartilages using same CT imaging procedure above.
[0219] 3. Calculate mean attenuation of every slice in cartilage
stack using ImageJ software: (i) Draw a region of interest (ROI) of
cartilage and keep ROI same between different cartilage slices.
(ii) Go to Image.fwdarw.Adjust.fwdarw.Threshold and set range as
-300 to +.infin.. Any intensity below -300 will be set to `NaN`.
(iii) Go to Image.fwdarw.Stacks.fwdarw.Measure Stack to acquire the
mean attenuation of each slice. (iv) The average of the mean
attenuation of all the cartilage slices is used as mean CECT
attenuation of cartilage. [0220] 4. After CT imaging, digest each
cartilage using 1 mL of 1 mg/mL protease K for 48 h at 57.degree.
C. Vortex each vial properly and measure the total residual
glycosaminoglycans (GAGs) in digested explants using the DMMB
assay. The GAG content (mg/mg wet weight) is calculated as:
[0220] GAG content = Total residual GAGs ( mg ) Wet weight of
cartilage ( mg ) ##EQU00001## [0221] 5. Build up the correlation
graph between CECT mean attenuation and GAG content in cartilage
(total GAG concentration per unit wet weight of cartilage) as
showed in FIGS. 10A and B. A strong positive correlation between
mAv-IOX CECT attenuation and residual GAG content in cartilage
explant should be observed indicating the high potential of
cationic contrast agent, mAv-IOX, in diagnosing different stages of
OA.
Analysis
Degree of Biotinylation Using HABA Colorimetric Assay
[0222] The extent of biotinylation of PEG and the loading of
PEG-biotin on Avidin were determined by using the HABA colorimetric
assay [5]. Changes in absorbance of the HABA-Avidin complex at 500
nm due to competitive displacement by the biotinylated PEG was used
to estimate the degree of biotinylation. HABA dye was dissolved in
10 mL of nanopure water (2.42 mg/mL) and filtered using 0.2 .mu.m
filter. Excessive HABA dye was added to Avidin solution to a final
concentration of 0.82 mg/mL (initial absorbance of 1.2). 20 .mu.L
of graded concentrations of PEG-biotin or Dex-PEG-biotin were added
to 180 .mu.L of HABA-Avidin complex (1:1 through 8:1 molar ratio of
PEG-biotin to HABA-Avidin) that competitively displaced HABA from
the biotin binding sites of Avidin thereby reducing the absorbance
value. 100% PEGylation of Avidin was achieved when the change in
absorbance achieved a plateau.
Gel Electrophoresis
[0223] Conjugation of PEG-biotin to Avidin was confirmed by using
native polyacrylamide gel electrophoresis (PAGE) in 7.5% separating
gel. In brief, 12 .mu.L of protein samples (.about.7.5 .mu.g
protein) in DI water were mixed with 4 .mu.L of 2.times. Native
Tris-Glycine sample loading buffer without heating. Since the
isoelectric point of Avidin is 10.5 and the protein mobility
depends on both the charge and molecular weight in the native PAGE
gel, the electrode polarity had to be reversed (anode was inserted
at the top of gel and cathode was inserted at the bottom of gel).
Electrophoresis was performed for approximate 4 h in 1.times.
solution of non-sodium dodecyl sulfate tris-base running buffer at
200 V, 40 mA and 4.degree. C.
[0224] Native gel was stained using iodine solution and Coomassie
Brilliant Blue R-250. Gel was fixed and then washed with deionized
(DI) water for 20 min. Gel was then incubated in 5% barium chloride
solution for 15 min followed by 3 washes in DI water. Subsequently,
the gel was stained with potassium iodide and iodine solution for 5
min to identify free or conjugated PEG. Following this, the gel was
stained with Coomassie Brilliant Blue R-250 for Avidin, and
de-stained three times in 100 mL of 10% acetic acid solution for 1
h.
Ultra Performance Liquid Chromatography (UPLC) and Zeta
Potential
[0225] PEGylation of Avidin in 1:2 and 1:4 mAv was further
confirmed by using H-Class Acquity UPLC (Waters Corp, Milford,
Mass.) equipped with an Acquity UPLC BEH200 Size Exclusion Column
(200 .ANG., 1.7 .mu.m column, 4.6.times.300 mm) with 20 mM ammonium
bicarbonate buffer as the mobile phase at 0.2 mL/min. Avidin was
detected at 280 nm. Zeta potential of Avidin and mAv was measured
in nanopure water at 0.45 mg/mL concentration using a Zetasizer
Nano-ZS90.
Proton Nuclear Magnetic Resonance (.sup.1H-NMR)
[0226] Modification of Dex and conjugation of Dex-SA, Dex-GA,
Dex-PA to PEG-biotin were verified using 500 MHz .sup.1H-NMR
(Varian Inova. Agilent Technologies). 1-2 mg of solutes to be
tested were dissolved in 700 .mu.L DMSO-d6. In addition,
.sup.1H-NMR spectra of Dex-SA, Dex-GA, Dex-PA reacted with
PEG-biotin using EDC/NHS chemistry were also confirmed. The
obtained NMR data was analyzed using MestRe Nova software.
Dex Loading Content
[0227] Dex-PEG-biotin was hydrolyzed using 0.1 N hydrochloric acid
overnight and neutralized against 0.1 N sodium hydroxide. The
amount of Dex released was quantified by HPLC (Agilent Technologies
1260 infinity II) equipped with a Variable Wavelength Detector
using a Poroshell 120 EC-C18 4.6.times.150 mm column. A gradient of
solvent A (0.1% trifluoroacetic acid (TFA) in water) and solvent B
(0.1% TFA in acetonitrile) was used. The concentration of solvent B
was increased linearly from 5% to 65% over 15 min. Column
temperature of 30.degree. C. and a flow rate of 1.0 mL/min were
used. Dex was detected at 254 nm. Drug loading content (DLC) was
calculated as:
DLC = Total Dex encapsulated ( g ) Total Dex encapsulated ( g ) +
Mass of Avidin ( g ) ( 1 ) ##EQU00002##
In-Vitro Dex Release Rates
[0228] Dex release rates from Dex-PEG-biotin were estimated in PBS
at pH 7.4, 37.degree. C. using dialysis tubing (7.0 kDa MWCO) with
continuous shaking under sink conditions: Dex concentration was
kept 10.times. lower than the saturation solubility of Dex in PBS.
At different time intervals, 200 .mu.L of release media was used to
estimate the Dex concentration by HPLC, which was replaced by equal
amount of fresh release media.
Transport Studies
Equilibrium Uptake of mAv in Cartilage
[0229] Cartilage explants were harvested from the femoropatellar
groove of 2-3-week-old bovine knees (Research 87, Boylston, Mass.)
with a 3 mm diameter biopsy punch. The cylindrical plugs were then
sliced to obtain the superficial 1 mm layer of cartilage, and
frozen until use. Dual labeled mAv was synthesized by conjugating
Texas Red labeled Avidin with FITC labeled 8-arm PEG. Cartilage
disks were equilibrated in 300 .mu.L of 8.5 .mu.M of labeled
Avidin, 1:2 mAv or 1:4 mAv in presence of protease inhibitors for
24 h in a 96-well plate at 37.degree. C. on gentle shaking to
prevent formation of stagnant layers. To minimize evaporation, the
empty wells in the plate were filled with DI water and the plate
was wrapped in parafilm. At the end of the experiment, the surfaces
of each disk were quickly blotted with Kimwipes and the wet weight
was measured. The equilibrium bath fluorescence was measured using
a plate reader (Synergy H1, Biotek). The final concentration was
calculated based on a linear calibration correlating fluorescence
to concentration of labeled Avidin. The moles of solutes absorbed
into the cartilage were calculated using the difference between the
initial and equilibrium concentration of the bath. The
concentration of solutes inside was calculated by normalizing the
number of moles inside cartilage to the wet weight of the tissue.
The uptake ratio (R.sub.U) was defined as the ratio of the
concentration of solutes inside the tissue (C.sub.tissue) to that
of the solute in the equilibrium bath (C.sub.bath).
R U = C tissue C b a t h ( 2 ) ##EQU00003##
[0230] To measure uptake of labeled Avidin and mAv in
partially-degraded tissue modeling osteoarthritic cartilage,
cartilage explants were incubated in 0.10 mg/mL trypsin-EDTA phenol
red solution in PBS for 5 h, which induced about 50% GAG depletion
[1]. GAG content in cartilage was measured using the
dimethyl-methylene blue (DMMB) assay [6]. The explants were then
rinsed multiple times and incubated in PBS containing protease
inhibitors for 1 h to wash out trypsin before using for uptake
experiments.
Confocal Imaging to Estimate Depth of Penetration into
Cartilage
[0231] A previously described transport setup was used to study
1-dimensional diffusion of solutes in cartilage. Briefly, 6 mm half
disk cartilage explants were mounted in the mid-section of the
chamber. The upstream compartment was filled with either 9.5 .mu.M
of labeled Avidin or dual labeled 1:2 and 1:4 mAv. The transport
chamber was placed in a petri-dish containing water to minimize
evaporation and placed on a shaker inside an incubator at
37.degree. C. After 24 h of adsorption, a 100 .mu.m slice was cut
from the center of the explant and imaged using a confocal
microscope (Zeiss LSM 700). Texas Red was excited using 555 nm
laser line and FITC was separately excited using 488 nm laser line.
Z-stack multilayer image of both channels (Red and Green) were
obtained to visualize distribution of mAv conjugates. The maximum
intensity of each channel was projected to the Z-axis.
Intra-Cartilage Diffusion Kinetics
[0232] A custom designed transport chamber made out of transparent
poly (methyl methacrylate) was used to measure non-equilibrium
one-dimensional diffusion of solutes in cartilage as described
previously [1]. The interior walls of the transport chamber were
equilibrated in 0.5% nonfat-dried bovine milk solution in PBS for
15 min to lessen non-specific binding of solutes to inner walls of
the transport chamber. Subsequently, the compartments of chamber
were rinsed with DI water. 3 .mu.M solution of labeled Avidin or
mAv was added to the upstream chamber. A 6 mm diameter cartilage
disk of 400-600 .mu.m thickness was placed between the upstream and
downstream compartments. Concentration of fluorescently labeled
solutes in the downstream chamber was measured over time by
exciting the downstream solution using a 480 nm laser line and
detecting the emission. Non-equilibrium diffusion curves were
thereby obtained by plotting normalized downstream concentration
(C.sub.D) to upstream concentration (Cu) over time. Effective
Diffusivity (D.sub.EFF), which is the diffusivity of solutes in
cartilage while binding interactions exist within the tissue was
estimated as follows:
.tau. Lag = L 2 6 D E F F ( 3 ) ##EQU00004##
where L corresponds to cartilage thickness, and .tau..sub.Lag is
the time it takes to reach a steady state flux. .tau..sub.Lag was
estimated using the time-axis intercept of the linear slope of the
normalized concentration versus time.
[0233] In-vitro Cartilage Explant Culture Model of OA 3 mm diameter
cartilage explants harvested from calf knee joints were
equilibrated separately in serum free culture media containing
96.2% low-glucose DMEM, 1.0% HEPES, 1.0% NEAA, 1.0% PSA, 0.4%
proline and 0.4% ascorbic acid for 48 h at 37.degree. C., 5%
CO.sub.2 for 2 days prior to treatment. Tissue explants for all
treatment conditions were matched for depth and location to prevent
any bias. To test the biological effectiveness of mAv-Dex,
equilibrated cartilage explants were treated with or without
IL-1.alpha. (2 ng/mL) for 16 days in combination with (i) a single
dose of 100 nM free Dex, (ii) a continuous dose of 100 nM free Dex,
(iii) a single dose of 10 .mu.M free Dex (iv) a single dose of fast
release mAv-Dex (10 .mu.M Dex) or (v) a single dose of controlled
release mAv-Dex (2:1:1) (10 .mu.M Dex). To evaluate the effect of
mAv carrier alone on cartilage health, a high one-time dose of 10
.mu.M mAv, which is 10.times. higher than that used to deliver 10
.mu.M Dex in condition (v) above, was also tested in the absence of
IL-1.alpha.. Note that mAv here refers to 1:4 mAv configuration.
Media was changed every 2 days and IL-1.alpha. was replenished at
each medium change. Single dose treated explants were subjected to
the drug and its carrier for only the first 2 days; in the
following media changes, the media did not contain the drug,
thereby simulating a single intra-articular injection in-vivo [4].
In the continuous dosing condition, Dex was replenished throughout
the culture duration. IL-1.alpha. concentration was chosen as it
represents a moderately aggressive cytokine treatment. Previous
work has shown that a sustained (continuous) dosing of 10-100 nM
Dex throughout the culture duration is effective in suppressing
cytokine induced catabolism [4]; we, therefore, compared single and
continuous dosing with 100 nM free Dex. Single dose of mAv-Dex with
effective Dex concentration of 10 .mu.M was chosen for
intra-cartilage drug depot delivery to provide a sustained drug
dose of at least 10 nM Dex throughout the culture duration and
compared with the equivalent concentration of free Dex.
GAG Loss and Nitrite Release from Cartilage
[0234] After 16 days of culture, cartilage explants were weighed
and digested in proteinase K. The cumulative GAGs released to the
media and residual GAGs in the digested explants were measured
using the DMMB assay [6]. Nitrite content was measured using the
Griess assay as an indicator of nitric oxide (NO) release from
tissues. Equal volumes of Griess reagent and culture media
collected every two days were mixed and incubated at room
temperature for 15 min, and absorbance at 540 nm was measured using
a plate reader. Sodium nitrite was used as a standard.
Chondrocyte Viability and Metabolism
[0235] Chondrocyte viability was analyzed by staining cartilage
explants with FDA (4.0 mg/mL) and PI (10.0 mg/mL) for live (green)
and dead (red) cells, respectively. Slices were washed with PBS and
imaged at 4.times. magnification (Nikon Eclipse Ts2R). The live and
dead images were overlaid using ImageJ. At the end of the culture,
tissue explants were incubated with media containing 1.times.
resazurin sodium salt (alamarBlue assay) for 3 h in dark at
37.degree. C. and 5% CO.sub.2. Cell metabolic activity was
estimated by measuring fluorescence at 530 nm excitation and 590 nm
emission wavelengths.
Histology and Immunohistochemistry
[0236] Cartilage explants were fixed in 4% formalin, embedded in
0.75% agarose for ease of handling, dehydrated in a graded series
of ethanol and xylenes, and embedded in paraffin. Transverse
sections (to obtain full thickness cartilage slices with
superficial and deep zones) were taken at 5 .mu.m thickness. These
sections were stained with 0.5% Safranin 0, 0.02% Fast Green and
Weigert's iron hematoxylin for GAG detection. Adjacent sections
underwent antigen retrieval using 0.1% hyaluronidase, 0.1% pronase
in PBS for 30 min at 37.degree. C., then were immunostained for
type II collagen using 1 .mu.g/mL mouse IgG1 (clone II-II6B3) or
normal mouse IgG1 as isotype control. Antibody detection was
performed using a VectaStain Elite ABC kit with
3,3-diaminobenzidine staining. Stained sections were imaged using a
Zeiss Axioplan2 equipped with an AxioCam HRc camera.
Statistical Analysis
[0237] Data is presented as Mean.+-.Standard Deviation. For all
studies, n=6-8 explants per condition and experiments were repeated
using explants from at least 3 animals. A general linear mixed
effects model was used with animal as a random variable. For
comparisons between different treatment conditions, Tukey's
Honestly Significant Difference test was used. P<0.05 was
considered statistically significant.
Results
Synthesis and Characterization of mAv
[0238] The 1:5 molar ratio of PEG to NHS-biotin was optimal for
synthesizing biotinylated 8-arm PEG using NHS ester reaction
chemistry, resulting in every molecule of PEG being conjugated with
one molecule of biotin as confirmed by matrix-assisted
laser-desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS) (FIG. 11A-11B); PEG MW was estimated as 10620 Da
which increased to 10902 Da following biotinylation showing that on
an average 1.15 biotin per PEG were present. In addition to the
mass spectrometry data, degree of biotinylation was also confirmed
by using the HABA dye assay where the addition of biotinylated PEG
to the HABA-Avidin complex displaced the HABA dye and reduced the
absorbance value. Using the Beer-Lambert Law [5], an average of
1.28.+-.0.02 biotin per PEG molecule was estimated, which is
consistent with the mass spectrometry data.
[0239] PEGylation in mAv was confirmed using the HABA dye assay,
gel electrophoresis and UPLC. FIG. 2A shows the reduction in
absorbance value with increasing molar ratio of biotinylated PEG to
HABA-Avidin from 1:1 to 4:1, following which a plateau is achieved
meaning that a majority of biotin sites on Avidin were occupied by
PEG-biotin indicating the formation of 1:4 mAv. FIG. 2B shows
native PAGE gel in reverse polarity used to confirm PEGylation in
1:2 and 1:4 mAv containing two or four 8-arm PEGs, respectively.
PEG is stained as yellow with iodine and protein is stained as blue
with Coomassie Brilliant Blue R-250. In PEG-staining (left), bands
only appear in the PEG and mAv channels. However, in
protein-staining (right), bands only appear in the Avidin and mAv
channels. Therefore, bands at the same position in the mAv channels
with both PEG-staining and protein-staining verified the formation
of mAv.
[0240] UPLC also confirmed that a majority of the population in 1:4
mAv had 4 PEGs conjugated to Avidin (peak `b` at 4.38 min) followed
by a secondary population of mAv with 2 PEGs (peak `c` at 5.33 min,
FIG. 2C). No peak for native Avidin (6.29 min) was found in 1:4 mAv
confirming that Avidin was successfully PEGylated. UPLC of 1:2 mAv
also confirmed that the majority of Avidin conjugated with two PEGs
(Fig K1, peak `c` at 5.35 min) followed by configurations
containing three PEGs (peak `b`) and one PEG (peak `d`). Due to
this heterogeneity, the physical and transport properties
determined, represent the collective behavior of different
populations in each formulation.
[0241] PEGylation did not reduce mAv's zeta potential (4)
suggesting minimum shielding of cationic charge; its net size was
within the 10 nm limit (Table 1, below). As such, its
intra-cartilage transport diffusivities (estimated using setup in
FIG. 3A) were not affected either (Table 1). The hydrodynamic
diameter of Avidin and mAv was estimated from their molecular
weights using the Stokes-Einstein equation. The net size of mAv was
within the 10 nm limit enabling it to penetrate through the full
thickness of cartilage similar to unmodified Avidin.
TABLE-US-00003 TABLE 1 Zeta potential (.zeta.), net size (diameter)
and intra-cartilage effective diffusivities (D.sub.EFF) of Avidin,
1:2 mAv and 1:4 mAv. Data shown as Mean .+-. SD. Formulation Avidin
1:2 mAv 1:4 mAv .zeta. (mV) 18.3 .+-. 0.5 20.3 .+-. 0.3 25.3 .+-.
0.7 Diameter (nm) ~7.0 ~7.6 ~8.1 D.sub.EFF (cm.sup.2/s) 8.4 .+-.
2.0 .times. 10.sup.-8 2.7 .+-. 0.5 .times. 10.sup.-8 3.5 .+-. 1.0
.times. 10.sup.-8
Transport Properties of mAv in Cartilage
[0242] Non-equilibrium transport properties of mAv in cartilage
were estimated from diffusion curves (FIG. 3A) using a setup
described previously [1]. Effective diffusivity (D.sub.EFF) of mAv
decreased 2.5-3.times. compared to unmodified Avidin, implying
slower diffusion rates as a result of PEGylation. There was no
significant difference in diffusivities of 1:2 and 1:4 mAv (Table
1).
[0243] Equilibrium intra-cartilage uptake (R.sub.U) of 1:2 mAv and
1:4 mAv was 1.5.times. and 1.3.times. lower compared to Avidin in
healthy cartilage, respectively (FIG. 3B), however, mAv still
maintained high uptake in cartilage (mean R.sub.U of 96 and 112 for
1:2 mAv and 1:4 mAv, respectively). Furthermore, following 50% GAG
depletion to simulate a mid-stage OA condition, as expected, uptake
of Avidin, 1:2 mAv and 1:4 mAv dropped by 13.2.times., 8.5.times.
and 4.8.times., respectively, compared to healthy condition due to
the loss of negatively charged GAG binding sites (FIG. 3B). It
should, however, be noted that these uptake values are still very
high. For example, a mean R.sub.U of 33 for 1:4 mAv implies
33.times. higher concentration inside cartilage than in the
surrounding equilibration bath. 1:4 mAv carriers can, therefore,
also be used for targeting early to mid-stage OA cartilage.
Additionally, a majority of mAv was retained inside healthy
cartilage over at least a 7-day period similar to unmodified Avidin
when desorbed in 1.times. PBS. Desorption in saline bath with high
salt concentration (10.times.PBS) resulted in complete desorption
for all species within 24 h highlighting the dominant role of
charge interactions (FIG. 3C).
[0244] 1:4 mAv penetrated through the full thickness of cartilage
within 24 h (FIG. 3D), similar to Avidin in both healthy and OA
cartilage explants. The green channel shows presence of PEG-FITC
while the red channel shows Avidin-Texas Red distribution in
cartilage.
Synthesis and Characterization of PEG-Dex-SA, PEG-Dex-GA,
PEG-Dex-PA
[0245] Incorporation of carboxyl group in compounds 2, 3 and 4
(FIG. 4A) was verified with the appearance of a yellow spot
(stained by bromocresol green) on TLC plate and their yields were
estimated as .about.92%, .about.72% and -94%, respectively, using
HPLC quantitative analysis. PEG-Dex-SA (5), PEG-Dex-GA (6) and
PEG-Dex-PA (7) were synthesized by coupling compounds 2, 3 or 4
with 8-arm PEG-amine through EDC/NHS reaction (FIG. 4A). The
chemical structure of compounds 1-7 were confirmed by .sup.1H-NMR
as shown in FIG. 13A-F.
[0246] The amount of Dex conjugated to PEG was determined by
analytical reverse-phase HPLC. As shown in Table 2, below,
compounds 5, 6 and 7 had 6.6.+-.0.5, 1.6.+-.0.4, 3.3.+-.0.5
molecules of Dex on one molecule 8-arm PEG, respectively. After
conjugating to Avidin in 1:4 mAv configuration, the DLC for
mAv-Dex-SA, mAv-Dex-GA, and mAv-Dex-PA were calculated as
15.7.+-.1.0%, 3.8.+-.0.9%, and 7.8.+-.0.1%, respectively.
TABLE-US-00004 TABLE 2 Hydrolysis half-lives of ester linkers
between 8-arm PEG and carboxylated derivatives of Dex. Molar ratio
of Dex conjugated with PEG for each configuration and the
corresponding drug loading content (DLC). Data shown as Mean .+-.
SD. PEG-Dex Ester linker PEG-Dex-SA PEG-Dex-GA PEG-Dex-PA (2:1:1)
Half-life (h) 6.8 .+-. 0.2 79 .+-. 1.8 86 .+-. 2.3 38.5 .+-. 1.5
Dex:PEG 6.6 .+-. 0.5:1 1.6 .+-. 0.4:1 3.3 .+-. 0.5:1 4.6 .+-. 0.5:1
(molar ratio) DLC (%) 15.70 .+-. 1.0 3.81 .+-. 0.9 7.85 .+-. 0.1
11.1 .+-. 0.8
[0247] About 70% of Dex was released from PEG-Dex-SA in PBS within
the first 24 h resulting in a short release half-life (t.sub.1/2)
of about 6.8 h (FIG. 4B). We addressed this by increasing the
carbon spacer length between the ester and adjacent amide by
replacing SA with GA or PA to form carboxylic acid derivatives of
Dex, which significantly increased the mean release half-lives to
79 h and 86 h for PEG-Dex-GA and PEG-Dex-PA, respectively. These
spacers weaken the adjacent carbonyl groups' inductive effect
(which reduces the ester's electron density) by donating multiple
electrons and stabilizing the ester bond (FIG. 4C). These linkers
provide a simple aqueous based way of effectively controlling
release rates depending on drugs, disease severity and tissue type.
Based on the drug release profiles, compounds 5, 6 and 7 were
physically mixed at a 2:1:1 molar ratio by Dex content (PEG-Dex
2:1:1) to develop a controlled release formulation that combined
the effects of burst-release from PEG-Dex-SA to reach therapeutic
levels within a short period of time and sustained drug release
over two weeks from PEG-Dex-GA and PEG-Dex-PA. The resulting Dex
release half-life was measured as t.sub.1/2=38.5.+-.1.5 h. In vivo
release half-lives of linkers are expected to be altered due to the
presence of proteases, other enzymes and binding with other
synovial fluid proteins. Prior work, however, has shown that Dex
release via ester hydrolysis did not change in synovial fluid and
was similar to that in PBS [4].
[0248] Finally, before conjugating PEG-Dex-SA to Avidin to form
fast release mAv-Dex or a combination of PEG-Dex-SA, -GA and -PA in
2:1:1 molar ratio to Avidin to form controlled release mAv-Dex
(2:1:1) and testing its bioactivity using cytokine challenged
in-vitro cartilage explant culture models, cartilage explants were
treated with compounds 5, 6 and 7 (with high equivalent
concentration of 100 .mu.M Dex, which is 10.times. higher than that
used in subsequent bioactivity experiments) for 48 h. Treated
explants showed similar cell viability as the untreated control
condition (FIG. 4D), confirming no cytotoxic effects.
Bioactivity of Single Dose of mAv-Dex in IL-1.alpha. Treated
Cartilage Explant Culture OA Model
[0249] We first compared the effectiveness of single versus
continuous (sustained) doses of 100 nM free Dex in suppressing
IL-1.alpha. induced catabolic activity in cartilage. Treatment with
IL-1.alpha. significantly increased GAG loss over the 16-day
culture period compared to control (p<0.0001) (FIG. 5A). A
single dose of 100 nM Dex was not effective and a continuous dose
of Dex was needed to effectively suppress IL-1.alpha. induced GAG
loss throughout the culture duration to levels similar to the
healthy condition, highlighting the importance of maintaining a
sustained drug concentration throughout the culture period.
[0250] To evaluate the effect of single dose of mAv-Dex that
creates an intra-cartilage drug depot, a concentration of 10 .mu.M
Dex was used to provide sustained therapeutic doses of at least 10
nM over the entire culture duration and the biological effects were
compared with that of free Dex. The dosing calculations are
explained here: Previous work has shown that a sustained
(continuous) dosing of 10-100 nM Dex throughout the culture
duration is effective in suppressing cytokine induced catabolism
[5, 6], which corresponds to a minimum of 0.0001-0.001 nmol inside
cartilage, respectively. An initial dose of 10 .mu.M mAv-Dex in the
media equilibrates within 24 h resulting in 112.times. higher
concentration inside cartilage than the outside media bath owing to
charge interactions (FIG. 3B). Therefore, based on mass balance
between the explant and surrounding media, the equilibrium Dex
concentration in media is estimated as 2.1 .mu.M, implying an
intra-cartilage concentration of -236 .mu.M (112.times.2.1 .mu.M),
which is equivalent to 2.36 nmol of Dex inside the cartilage disk
of 10 .mu.L volume before the first media change at 48 h. Dex
release half-life from mAv-Dex in 2:1:1 formulation is 38.5 h (by
fitting a single-phase exponential decay model), which means 10
half-lives over 16-day culture period. Based on this,
[(1/2){circumflex over ( )}10] of the initial administered dose
will be present in cartilage after 16 days which equals to 0.0023
nmol (0.1% of 2.36 nmol). Therefore, a one-time dose of 10 .mu.M
mAv-Dex was used in our experiments as it can maintain therapeutic
Dex dosage of at least 10 nM over 2 weeks to suppress cartilage
degradation.
[0251] As shown in FIG. 5B, a single dose of 10 .mu.M Dex
significantly suppressed IL-1.alpha. induced GAG loss throughout
the duration of culture (p<0.0001 compared to IL-1.alpha.
condition) but remained significantly elevated compared to the
control condition (p<0.0014 starting at Day 4), highlighting its
short-term therapeutic benefit. Both fast and controlled release
mAv-Dex suppressed IL-1.alpha. induced GAG loss throughout the
culture duration significantly greater than single Dex dose (from
Day 4; p<0.0037), bringing levels down to that of the untreated
control. Controlled release mAv-Dex (2:1:1) had a longer lasting
therapeutic response (throughout the culture duration) than the
fast release mAv-Dex whose GAG loss levels became statistically
different from control condition by Day 8 (p<0.04). mAv carrier
in 10.times. higher concentrations than used in mAv-Dex (2:1:1) was
associated with no GAG loss. In fact, GAG loss levels measured were
lower than that for the control condition, a phenomenon that
warrants more probing. We confirmed that the presence of positively
charged Avidin did not hinder the activity of cationic DMMB assay
as the total GAG measured (i.e. GAG lost to media over the entire
culture duration+residual GAG in the cartilage explant) was similar
in the untreated control and mAv treated conditions i.e. we were
effectively able to measure the total GAG content using the DMMB
assay.
[0252] Similar trends were observed in Day 16 chondrocyte viability
images (FIG. 5C); treatment with IL-1.alpha. enhanced cell death
which was rescued effectively by the continuous dose but not with a
single dose of 100 nM Dex. On the other hand, single dose of both
fast and controlled release 10 .mu.M mAv-Dex rescued cell viability
similar to continuous dosing condition while a single dose of 10
.mu.M free Dex was not as effective. mAv treatment did not cause
any cell death. Some cell death shown in the superficial zone was
observed in control explants, along with the location of harvesting
from the joint. Additionally, excision of tissues from the joint
using punches can also lead to cell death at the cut surfaces.
[0253] Nitrites are a reactive oxygen species (ROS) triggered by an
upregulation in inflammatory activity such as the presence of
IL-1.alpha. in an OA environment. As expected, IL-1.alpha. treated
explants produced 14.4.times. and 7.7.times. higher amounts of
nitrite compared to control at 2 and 8 days of culture
(p<0.0001), respectively (FIG. 6A). Treatment with all Dex
containing conditions significantly lowered nitrite synthesis at
Day 2 (p<0.047 vs IL-1). By Day 8, continuous Dex dose resulted
in 3.5.times. lower nitrite levels compared to single dose of 100
nM Dex. A single dose of mAv-Dex (both controlled and fast release
formulations) suppressed nitrite synthesis levels by 1.5-2.times.
compared to a single dose of 10 .mu.M free Dex, bringing levels
close to control levels at Day 8. Lastly, there was no difference
between mAv and untreated control conditions confirming no
associated toxicity/inflammatory effects of mAv. IL-1.alpha.
reduced cell metabolism rates, however, were not rescued by either
Dex or mAv-Dex (FIG. 6B) suggesting the need to incorporate a
pro-anabolic drug for a comprehensive and a more effective OA
treatment. mAv treatment did not suppress cell metabolism
levels.
[0254] Finally, GAG and collagen content through the explant tissue
depth was assessed by histology and immunohistochemistry (FIG. 7).
IL-1.alpha. treated explants showed visibly low levels of
Safranin-O staining compared to controls, indicating significant
GAG loss. This IL-1-stimulated GAG loss was mostly rescued by a
continuous 100 nM Dex dose, particularly below the superficial
layer where chondrocyte viability was maintained (FIG. 5A-C);
however, staining was marginally affected by a single 100 nM dose.
At 10 .mu.M, a single dose free Dex protected GAG content to a
greater extent than at 100 nM. The fast release formulation of 10
.mu.M mAv-Dex did not clearly alter GAG staining over 10 .mu.M free
Dex. This may be partly explained by the limited linear range of
GAG staining by Safranin-O, which can mask more subtle differences
in total GAG levels; in this regard, Safranin-O staining can better
reflect the localization of GAG loss. In contrast to the fast
release formulation, the controlled release mAv-Dex formulation
displayed GAG staining that more closely approximated control disks
compared to the other IL-1-challenged groups. Furthermore, mAv
alone did not have any detrimental effects on tissue Safranin-O
staining. Total type II collagen levels were not visibly affected
by IL-1.alpha. treatment over the 16 days test period (FIG. 7).
[0255] Discussion
[0256] Here we designed a cartilage penetrating and binding mAv
nano-construct that can be conjugated with a variety of small
molecule drugs or their combinations using hydrolysable ester
linkers with tunable drug release rates. The nano-construct
incorporates four 8-arm PEGs, providing 28 sites for drug
conjugation per mAv compared to only 4 sites in previous designs
[4]. As a result, to deliver a one-time dose of 10 .mu.M Dex to
cartilage explants, less than 1 .mu.M Avidin was needed which is
within safe limits. Previous research has shown that an Avidin
concentration less than 100 .mu.M does not cause any GAG loss or
affect chondrocyte viability or biosynthesis rates of proteins and
GAGs in bovine cartilage explants. mAv was designed to keep its net
size within the previously determined 10 nm size limit for
unhindered diffusion into native cartilage and its net charge was
shown to be not shielded by addition of multi-arm PEGs (Table 1).
As a result, mAv exhibited similar intra-cartilage transport
properties as native Avidin; it penetrated through the full
thickness of cartilage within 24 h, resulting in a high
intra-cartilage uptake (mean R.sub.U of 112 for 1:4 mAv) and
long-term retention as the majority of it remained bound within the
cartilage over a 7-day desorption period (the duration of
experiment conducted) in 1.times.PBS (FIG. 3A-D). mAv also showed
high uptake (mean R.sub.U of 33) in 50% GAG depleted cartilage
explants, implying that it can be used for delivering drugs to mid-
to late-stage arthritic cartilage. While some binding of mAv with
negatively charged groups of synovial fluid can be expected, our
previous work has confirmed that the high negative FCD of cartilage
enables rapid diffusion of IA injected native Avidin into rat and
rabbit cartilage [2,3] resulting in high uptake and long-term
retention despite the presence of synovial fluid and dynamic
compression-induced convective flow in animal knee joints. Since
mAv's net size and charge properties are similar to that of native
Avidin, similar intra-joint kinetics are expected.
[0257] First, we conjugated Dex as an example OA drug with 8-arm
PEG using hydrolysable ester linkers derived from SA (FIG. 4A).
However, the fast-release ester linker in PEG-Dex-SA had a
half-life of 6.8.+-.0.2 h, which burst-released a majority of Dex
prior to the first media change at 48 h of culture (FIG. 4B). When
conjugated with Avidin (fast release mAv-Dex), this resulted in
significantly improved therapeutic benefit compared to free Dex
throughout 16 days of culture. However, compared to continuous dose
of 100 nM Dex, the fast release mAv-Dex resulted in higher GAG loss
starting at Day 8 (FIG. 5B). Thus, in order to maintain therapeutic
doses of Dex throughout the culture duration, we used GA or PA to
synthesize carboxylated derivatives of Dex that increased the
half-life of the ester linkers in PEG-Dex-GA and PEG-Dex-PA to
79.+-.1.8 h and 86.+-.2.3 h, respectively. The hydrolysis of an
ester bond begins when hydroxide ions of water attack the
electrophilic carbon in the ester bond, breaking the p-.pi.
conjugation of ester bond creating a tetrahedral intermediate.
Since the adjacent carbonyl group from the amide bond tends to
compete with and withdraw electrons from the ester bond (inductive
effect) resulting in a decrease in the ester bond's electron
density, the carbon in ester bond becomes more electrophilic and
reactive to nucleophilic attack from hydroxide ion causing a faster
release rate (as in the case of compound 5, PEG-DEX-SA in FIG. 4C).
Furthermore, hydrophobic Dex conjugated to hydrophilic polymer PEG
can produce repulsive forces, thereby accelerating the separation
of Dex from PEG. As for compound 6, the GA cross-linker will
increase the carbon length between the ester and amide bonds thus
weakening the inductive effect of the carbonyl group. The two
methyl groups in the GA branched chain can also donate electrons,
thereby stabilizing the ester bond. Similarly, the phenyl group in
PA can donate numerous electrons to the ester bond (compound 7).
Our previous research has shown that a combination of fast and slow
releasing linkers can yield a more effective treatment over a long
period of time than a complex containing only one of the linkers
[4]. Thus, we used ester linkers derived from SA, GA and PA in a
molar ratio of 2:1:1 to formulate a controlled release mAv-Dex that
enabled 50% release of Dex in 38.5 h followed by a sustained
release of the remaining drug over the next two weeks.
[0258] As a result, single 10 .mu.M dose of controlled release
mAv-Dex (2:1:1) completely suppressed IL-1.alpha. induced GAG loss
bringing levels down to that of untreated control throughout the
16-day culture period (FIGS. 5B and 7), mimicking the continuous
100 nM Dex condition (FIGS. 5A and 7). Its therapeutic effect was
significantly better than that of the fast release mAv-Dex where
ester linker was derived from SA only and had a short half-life of
6.8 h. Similar trends were observed in Day 16 chondrocyte viability
images (FIG. 5C) where single dose of both fast and controlled
release 10 .mu.M mAv-Dex rescued cell viability similar to
continuous 100 nM free Dex while a single dose of 10 .mu.M free Dex
was not effective. Treatment with mAv alone did not have any
detrimental biological effect even at 10.times. higher
concentrations than used in mAv-Dex (2:1:1).
[0259] We chose Dex as an example drug as it is a broad spectrum
glucocorticoid (GC) with anti-catabolic properties and has
intra-cellular receptors inside cartilage and in synovium and can
elicit disease modifying effects as well as suppress OA induced
pain and inflammation. Effectiveness of GCs, however, has been a
subject of controversy showing both chondroprotective and
deleterious effects following IA injection. Their effectiveness
depends largely on frequency, dosage and the duration of treatment.
GCs like Dex, triamcinolone acetonide (TCA) and prednisone are used
in high doses and multiple times (up to 100 mM compared to one time
10 .mu.M dose used in the present study) for OA pain relief due to
their short intra-joint residence time causing bone resorption,
cell apoptosis and systemic toxicity. For example, a recent human
clinical trial (NCT01230424) concluded that IA injections of 40 mg
TCA every 3 months for 2 years in patients with symptomatic knee OA
resulted in greater cartilage loss compared to saline, emphasizing
the critical need for targeted, low dose sustained therapy. A
recent study also showed that multiple intra-articular injections
of Dex (2.5 mg every 3 days or 0.5 mg/kg) were needed to
significantly reduce inflammation and protect cartilage in a bone
drill rabbit model of PTOA; this high multi-dose regimen, however,
resulted in systemic toxicity in vital organs [7]. Nevertheless, a
recent clinical trial (NCT01692756) concluded that early
intervention using two doses of 40 mg TCA within 10 days of ACL
rupture was able to prevent injury induced early chondral changes.
While more follow up studies are needed to conclude meaningful
clinical difference in overall outcome, there remains continued
interest in using GCs as disease modifying agents for OA/PTOA
treatment and not just for symptomatic relief. It is, however,
imperative to develop strategies in parallel that can target
cartilage and deliver therapeutic low drug doses over several days
to weeks to avoid toxicity associated with multiple injections of
high drug doses.
[0260] Recent research has focused on developing IA drug delivery
systems, including drug-encapsulating microparticles, polymeric
micelles, liposomes, aggregating hydrogels and peptides. The
majority of carrier systems cannot penetrate the dense, negatively
charged cartilage; consequently, while some have shown promise for
suppressing pain and inflammation originating from the synovium and
surrounding joint capsule, they are not effective at stimulating
disease-modifying responses in chondrocytes. For example, recently
intra-joint sustained release formulations of TCA encapsulated
within micron sized PLGA particles (Flexion Therapeutics,
Burlington, Vt., USA) were approved by the FDA for OA pain and
inflammation, but such systems naturally use high drug doses to
induce biological response (40-60 mg of drug). Flexion's
microsphere based TCA delivery (FX006, 32 mg drug dose
administered) showed prolonged synovial fluid joint residency
(until Week 12), diminished peak plasma levels and thus reduced
systemic exposure compared to free TCA following a single IA
injection in patients with knee OA in a Phase II open label study.
Their Phase III, multicenter, double-blinded, 24-week study
concluded that a single IA injection of FX006 provided significant
improvement in average-daily-pain (ADP)-intensity scores compared
to saline (placebo) but no significant improvements in OA pain were
observed when compared to the free drug. In a Phase III post-hoc
study, where efficacy of FX006 was evaluated in a subgroup of
participants with unilateral knee OA only (as bilateral knee pain
has emerged as a cofounding factor in clinical trials evaluating
the effect of single IA injection), significant improvement in
WOMAC or ADP scores were reported for FX006 compared with both
saline and free drug over a period of 5-6 months. While these are
promising data for longer lasting pain relief with a single IA
injection, delivery systems like mAv can target cells inside
cartilage to elicit long-term disease modifying effect to restore
joint function. Joint inflammation has a complex etiology that
involves not only the synovium but also subchondral bone and
articular cartilage. Therefore, effective treatments will likely
need to deliver OA drugs into cartilage as well as to surrounding
tissues. We predict that gradual release of drug from
intra-cartilage drug depot enabled by mAv delivery can
significantly inhibit IL-1.alpha. signaling, for example, in both
cartilage and nearby synovium and other tissues inside the joint,
and thus can provide long-term pain and inflammation relief along
with restoring joint function. Future studies will investigate
this.
[0261] While a single dose of mAv-Dex effectively suppressed
cytokine-induced catabolic activity, it was unable to rescue
anabolic activity within cartilage explants (FIG. 6B). Dex is known
as a potential OA prophylactic agent that inhibits production of
matrix metalloproteinases (MMPs), NO and inflammatory cytokines in
OA joints. When combined with a pro-anabolic factor like
insulin-like growth factor 1 (IGF-1), it can effectively also
reverse cytokine-induced inhibition of GAG synthesis. Thus, a
combination drug therapy comprising of an anti-catabolic agent like
Dex that can induce broad spectrum inhibition of cytokine-related
degradation, maintain cell viability along with a pro-anabolic
factor like IGF-1 or Kartogenin that can stimulate biosynthesis of
viable chondrocytes and replenish cartilage matrix components like
aggrecans and collagens is a better approach than monotherapy with
one drug. Multiple drugs can be conjugated using similar chemistry
to mAv and their release rates can be modulated by varying the
ratio of ester linkers derived from SA, GA and PA or designing new
types of linkers based on the mechanism explained earlier.
[0262] Thus, by using charge interactions, our mAv nano-construct
offers multiple advantages of (i) converting cartilage from a
barrier to drug entry into a reservoir of drugs that can prevent
rapid exit from synovial joint, (ii) accumulating higher drug dose
at the cell and matrix target sites and (iii) presenting multiple
sites for covalent conjugation of more than one drug for
combination therapy. In summary, a charge-based cartilage homing
drug delivery platform like this can, potentially, elicit disease
modifying effects as well as facilitate long-term symptomatic pain
and inflammation relief by enhancing tissue specificity and
prolonging intra-cartilage residence time of OA drugs.
[0263] The mechanism of mAv targeting of cartilage and drug
delivery is summarized as follows (FIG. 1): Following its
intra-articular administration, mAv-Dex can rapidly penetrate
through the full thickness of negatively charged cartilage in high
concentrations due to electrostatic interactions thereby creating
an intra-cartilage drug depot. The optimal net positive charge of
mAv enables its rapid and high intra-cartilage uptake (112.times.)
and long-term retention via weak-reversible binding with negatively
charged aggrecans. Therapeutic doses of drug (Dex) is then released
via hydrolysis from controlled release mAv-Dex (2:1:1) over 2
weeks, which can diffuse into chondrocytes to bind with its
glucocorticoid receptors triggering downstream signaling pathways
and suppressing OA associated catabolic activity. This
intra-cartilage depot drug delivery platform can be used to deliver
a variety of drugs or combination of drugs and enable OA treatment
with only a single injection of low drug doses thereby eliminating
toxicity issues associated with multiple injections of high drug
doses that are currently needed to maintain sustained drug doses
within the joint.
Chemical Synthesis of CPC-IGF-1
[0264] Due to the highest intra-cartilage uptake, high
intra-cartilage retention and full-thickness penetration, CPC +14
sequence was chosen as an exemplary candidate for drug delivery to
cartilage. CPC +14 was conjugated to IGF-1 using maleimide
chemistry. Briefly, the fluorescently labeled CPC +14 was modified
with a lysine group at the C-terminus (CPC +14-K). CPC +14-K was
then modified with a maleimide group through reaction with
amine-reactive N-hydroxysuccinimide (NHS ester) bifunctional
maleimide linker,
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC) (see scheme above). Since all the cysteines in IGF-1
structure actively form disulfide bridges which makes them not
available for reaction with maleimide groups, reactive sulfhydryl
(SH) groups were added to lysine residues on IGF-1. For this aim,
SH groups were introduced to IGF-1 amine groups through reaction
with Traut's reagent to obtain IGF-1-SH. CPC-IGF-1 was subsequently
achieved through mixing of IGF-1-SH with CPC-Maleimide.
Chemical Synthesis of CPC-IOX
##STR00021##
[0266] CPC (+8) was conjugated to IOX through reaction of arginine
guanidinium group with an amine reactive succinimidyl carbonate
moiety introduced at hydroxyl functional group of IOX. For this
purpose, following (FIG. 6), 58.1 mg of IOX was dissolved in 1 mL
of DMF (46.0 .mu.mol, 1 equiv.). 58.8 mg of N,N'-Disuccinimidyl
carbonate (DSC) (229.0 .mu.mol, 5 equiv.) and 32.5 mg of
4-Dimethylaminopyridine (DMAP) (229.0 .mu.mol, 5 equiv.) were also
dissolved in 0.5 mL of DMF. Solutions of DSC and DMAP were slowly
added to IOX solution and reacted overnight at room temperature
under stirring. The IOX-succinimidyl carbonate (IOX-SC) conjugate
was precipitated using diethyl ether and collected for
lyophilization through centrifugation. "(RRAAA)3RR" disclosed as
SEQ ID NO: 5.
##STR00022##
[0267] There were no primary amines available in the CPC +8 to
react with IOX-SC group since the N-terminus of the peptide was
modified with 5-FAM. Therefore, the guanidinium groups of CPC +8
were deprotonated using Barton's base to facilitate their reaction
with the amine reactive succinimidyl carbonate introduced on IOX
following, similar to reaction between active esters and guanidine
groups of arginine. For this aim, 0.05 mg of CPC +8 was dissolved
in 0.05 ml of DMSO (20.2 .mu.mol, 1 equiv.) and 0.15 mg of IOX-SC
(106.5 .mu.mol, 5.3 equiv.) was dissolved in 0.05 ml of DMF. The
solutions were mixed and reacted at 40.degree. C. for 4 h under
constant shaking by addition of 1 .mu.l of Barton's base. The final
product was lyophilized and stored.
Cartilage Uptake of CPC-IOX
[0268] CPC +8-IOX demonstrated 63 times higher uptake ratio
compared to pristine IOX (FIG. 14A). Additionally, CPC +8-IOX
penetrated through the full thickness of cartilage within 24 h
(FIG. 14B). The 2D color maps demonstrated that a low 0.5 mgI/mL of
CPC +8-IOX produced high attenuation and spatial distribution
similar to 16 mgI/mL IOX. However, same concentration of IOX (0.5
mgI/mL did not induce any signal) (FIG. 14C). Therefore, upon
conjugation with CPC +8, the required concentration of IOX for
effective cartilage CT imaging attenuation was reduced by 32 times
owing to the electrostatic interactions between the CPC +8 modified
IOX with the negatively charged cartilage GAGs. Additionally,
attenuation of IOX was mostly at the superficial zone of cartilage
while CPC +8-IOX attenuation was the highest at the deep zone of
cartilage, which correlates directly with the higher density of
negatively charged GAG groups in the deep zone of cartilage.
Cartilage CT Imaging with mAv-IOX
[0269] FIG. 15 shows that mAv-IOX is significantly more effective
in producing a CT signal in cartilage explants with varying
arthritis severity, which was simulated by depleting the negatively
charged glycosaminoglycans (GAGs) from the tissue samples using an
enzyme. The CT signal correlated strongly with spatial distribution
of GAGs inside cartilage. A strong positive correlation was
observed between the mean CT attenuation using mAv-IOX, which was
not possible with IOX alone.
REFERENCES
[0270] 1. A. Vedadghavami, et al., Cartilage penetrating cationic
peptide carriers for applications in drug delivery to avascular
negatively charged tissues, Acta biomaterialia, 93 (2019)
258-269.10.1016/j.actbio.2018.12.004 [0271] 2. A. G. Bajpayee, et
al., A rabbit model demonstrates the influence of cartilage
thickness on intra-articular drug delivery and retention within
cartilage, J Orthop Res, 33 (2015) 660-667.10.1002/jor.22841 [0272]
3. A. G. Bajpayee, et al., Sustained intra-cartilage delivery of
low dose dexamethasone using a cationic carrier for treatment of
post traumatic osteoarthritis, Eur Cell Mater, 34 (2017)
341.10.22203/eCM.v034a21 [0273] 4. A. G. Bajpayee, et al., Charge
based intra-cartilage delivery of single dose dexamethasone using
Avidin nano-carriers suppresses cytokine-induced catabolism long
term, Osteoarthritis Cartilage, 24 (2016)
71-81.10.1016/j.joca.2015.07.010 [0274] 5. N. M. Green, A
Spectrophotometric Assay for Avidin and Biotin Based on Binding of
Dyes by Avidin, Biochem J, 94 (1965) 23C-24C.10.1042/bj0940023c
[0275] 6. R. W. Farndale, C. A. Sayers, A. J. Barrett, A direct
spectrophotometric microassay for sulfated glycosaminoglycans in
cartilage cultures, Connect Tissue Res, 9 (1982)
247-248.10.3109/03008208209160269 [0276] 7. K. D. Huebner, N. G.
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INCORPORATION BY REFERENCE
[0277] All of the U.S. patents and U.S. and PCT published patent
applications cited herein are hereby incorporated by reference.
EQUIVALENTS
[0278] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Sequence CWU 1
1
5120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala Ala
Arg Arg Ala Ala1 5 10 15Ala Ala Arg Arg 20220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Arg
Arg Arg Arg Ala Ala Arg Arg Arg Ala Ala Arg Arg Arg Ala Ala1 5 10
15Arg Arg Arg Arg 20332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Ala Arg Arg Arg Ala Ala
Arg Ala Ala Arg Arg Arg Ala Ala Arg Ala1 5 10 15Ala Arg Arg Arg Ala
Ala Arg Ala Ala Arg Arg Arg Ala Ala Arg Ala 20 25
30420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg 20517PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Arg
Arg Ala Ala Ala Arg Arg Ala Ala Ala Arg Arg Ala Ala Ala Arg1 5 10
15Arg
* * * * *