U.S. patent application number 11/060383 was filed with the patent office on 2005-08-04 for controlled delivery of therapeutic agents by insertable medical devices.
This patent application is currently assigned to Boston Scientific Scimed, Inc.. Invention is credited to Leong, Kam W., Li, Weiping, Mao, Hai-Quan.
Application Number | 20050169969 11/060383 |
Document ID | / |
Family ID | 22633301 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169969 |
Kind Code |
A1 |
Li, Weiping ; et
al. |
August 4, 2005 |
Controlled delivery of therapeutic agents by insertable medical
devices
Abstract
A medical device and method for transportation and release of a
therapeutic agent into a mammalian body are disclosed. The medical
device is coated with alternating layers of a negatively charged
therapeutic agent and a cationic polyelectrolyte, following a
controlled adsorption technique. The method is simple, with minimal
perturbation to the therapeutic agent and uses clinically
acceptable biopolymers such as human serum albumin. The amount of
the therapeutic agent that can be delivered by this technique is
optimized by the number of the layers of the therapeutic agent
adsorbed on the surface of medical device. There is a washing step
between alternate layers of the therapeutic agent and cationic
polyelectrolyte carrier, so that the amount of the therapeutic
agent on the insertable medical device represents the portion that
is stably entrapped and adsorbed on to the medical device. The
insertable medical device and method according to this invention
are capable of reproducibly delivering therapeutic agent to a site
in a mammalian body, and allow for a highly reproducible and
controllable release kinetics of the therapeutic agent.
Inventors: |
Li, Weiping; (Salt Lake
City, UT) ; Mao, Hai-Quan; (Singapore, SG) ;
Leong, Kam W.; (Ellicott City, MD) |
Correspondence
Address: |
KENYON & KENYON
1 BROADWAY
NEW YORK
NY
10004
US
|
Assignee: |
Boston Scientific Scimed,
Inc.
|
Family ID: |
22633301 |
Appl. No.: |
11/060383 |
Filed: |
February 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11060383 |
Feb 17, 2005 |
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09750779 |
Jan 2, 2001 |
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6899731 |
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60173743 |
Dec 30, 1999 |
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Current U.S.
Class: |
424/426 ;
424/93.2; 514/291; 514/449; 604/500 |
Current CPC
Class: |
A61L 2300/416 20130101;
A61L 2300/258 20130101; A61L 29/16 20130101; A61L 31/16 20130101;
A61L 2300/602 20130101; A61L 2300/802 20130101; A61L 2300/608
20130101 |
Class at
Publication: |
424/426 ;
424/093.2; 514/291; 514/449; 604/500 |
International
Class: |
A61K 048/00; A61M
031/00; A61K 031/4745; A61K 031/337 |
Claims
1-46. (canceled)
47. A method of treating or preventing the course of a disease or
tissue dysfunction comprising: providing a medical device
comprising: an inner layer of a cationic polyelectrolyte carrier; a
layer of a negatively charged therapeutic agent adsorbed onto said
inner layer of a cationic polyelectrolyte carrier; and an
additional layer of a cationic polyelectrolyte carrier and an
additional layer of a negatively charged therapeutic agent adsorbed
onto said additional layer of said cationic polyelectrolyte
carrier, wherein said additional layer of a cationic
polyelectrolyte carrier and said additional layer of a negatively
charged therapeutic agent alternate; implanting the medical device
into a target location from which the negatively charged
therapeutic agent can treat or prevent the course of the disease or
tissue dysfunction.
48. The method of claim 47, wherein treating or preventing tissue
dysfunction comprises inhibiting angiogenesis.
49. The method of claim 48, wherein treating or preventing tissue
dysfunction comprises inducing angiogenesis.
50. The method of claim 49, wherein treating or preventing the
course of a disease or tissue dysfunction comprises treating or
preventing restenosis; cardiomyopathy or other dysfunction of the
heart; or cystic fibrosis or other dysfunction of the lung.
51. The method of claim 47, wherein treating or preventing the
course of a disease or tissue dysfunction comprises treating or
inhibiting malignant cell proliferation.
52. The method of claim 47, wherein treating or preventing the
course of a disease or tissue dysfunction comprises inducing nerve,
blood vessel, or tissue regeneration in a tissue.
53. The method of claim 47, wherein the medical device is used in
angioplasty.
54. The method of claim 47, wherein the medical device further
comprises an outermost layer of a cationic polyelectrolyte carrier
which is the same or different from the inner or the additional
layer of a cationic polyelectrolyte carrier.
55. The method of claim 47, wherein the inner or the additional
layer of a cationic polyelectrolyte carrier comprises human serum
albumin, gelatin, chitosan, or a combination thereof.
56. The method of claim 47, wherein the medical device is a stent
or a catheter.
57. The method of claim 56, wherein the catheter is a balloon
catheter.
58. The method of claim 47, wherein the negatively charged
therapeutic agent is rapamycin or paclitaxel.
59. The method of claim 47, wherein the negatively charged
therapeutic agent comprises more than one negatively charged
therapeutic agent.
60. The method of claim 47, wherein the negatively charged
therapeutic agent is selected from the group consisting of a:
anti-thrombogenic protein, antioxidant compound, angiogenic
protein, agent which blocks smooth muscle cell proliferation,
anti-inflammatory agent, calcium entry blocker,
antineoplastic/antiproliferative/anti-mitotic compound,
anti-microbial compound, anesthetic agent, nitric oxide donor,
anti-coagulant, vascular cell growth promoting protein, vascular
cell growth protein inhibitor, vascular cell growth antibody
inhibitor, cholesterol lowering drug, vasodilating drug, protein
that protects against cell death, cell cycle CDK protein inhibitor,
anti-restenosis protein, agent for treating malignancies, bone
morphogenic protein, a polynucleotide encoding any of the above
named proteins or protein inhibitors, and a vector comprising a
polynucleotide encoding any of the above named proteins or protein
inhibitors.
61. A method of delivering a therapeutic agent to a target location
comprising providing a medical device comprising: an inner layer of
a cationic polyelectrolyte carrier; a layer of a negatively charged
therapeutic agent adsorbed onto said inner layer of a cationic
polyelectrolyte carrier; and an additional layer of a cationic
polyelectrolyte carrier and an additional layer of a negatively
charged therapeutic agent adsorbed onto said additional layer of
said cationic polyelectrolyte carrier, wherein said additional
layer of a cationic polyelectrolyte carrier and said additional
layer of a negatively charged therapeutic agent alternate;
implanting the medical device into a target location to deliver the
negatively charged therapeutic agent to the target location.
62. The method of claim 61, wherein the target location is a brain,
heart, liver, skeletal muscle, smooth muscle, kidney, bladder,
intestine, stomach, pancreas, ovary, prostate, cartilage, bone,
lung, blood vessel, ureter, urethra, or testes.
63. The method of claim 61, wherein the at least one negatively
charged therapeutic agent is selected from the group consisting of
a: anti-thrombogenic protein, antioxidant compound, angiogenic
protein, agent which blocks smooth muscle cell proliferation,
anti-inflammatory agent, calcium entry blocker,
antineoplastic/antiproliferative/anti-mitot- ic compound,
anti-microbial compound, anesthetic agent, nitric oxide donor,
anti-coagulant, vascular cell growth promoting protein, vascular
cell growth protein inhibitor, vascular cell growth antibody
inhibitor, cholesterol lowering drug, vasodilating drug, protein
that protects against cell death, cell cycle CDK protein inhibitor,
anti-restenosis protein, agent for treating malignancies, bone
morphogenic protein, a polynucleotide encoding any of the above
named proteins or protein inhibitors, and a vector comprising a
polynucleotide encoding any of the above named proteins or protein
inhibitors.
Description
BACKGROUND
[0001] 1. Field of the Invention
[0002] The present invention relates to the localized delivery of
negatively charged therapeutic agents, and more particularly to the
localized and controlled delivery of DNA absorbed to the surface of
insertable medical devices, in particular, balloon catheters or
stents.
[0003] 2. Background of the Invention
[0004] It is often desirable to administer drug agents at localized
sites within the body because the systemic administration of drug
agents treats the body as a whole even though the disease to be
treated may be localized. Various methods have been proposed for
such localized drug administration. For example, U.S. Pat. No.
5,304,121, which is incorporated herein by reference, discloses a
method of delivering water-soluble drugs to tissue at desired
locations of a body lumen wall. The method generally includes the
steps of impregnating a hydrogel polymer on a balloon catheter with
an aqueous drug solution, inserting the catheter into a blood
vessel to a desired location, and expanding the catheter balloon
against the surrounding tissue to allow the release of the
drug.
[0005] One potential drawback to conventional localized drug
administration is the uncontrolled manner at which the drug or drug
solution is released from the delivery device. It is often desired,
if not necessary, to control and/or lengthen the time period over
which the drug is released. For example, it might be advantageous
to lengthen the release time from seconds to minutes, or from
minutes to hours, days, or even weeks. Exceptionally long release
times as long as several months are often desired, for example,
where the drug is released from an implanted device such as a
stent. Moreover, it is often desired to control the release rate of
the drug over prolonged periods of time.
[0006] Gene therapy provides an alternative approach to combating
many intractable cardiovascular diseases. A site-specific delivery
of the genetic vectors to minimize systemic complications is
crucial for the therapeutic potential of this approach to be
realized. Advances in interventional radiology and innovative
designs in balloon angioplasty and stents have raised that
possibility.
[0007] The invention disclosed herein solves the potential
drawbacks to the drug delivery methods and instruments of the prior
art by providing novel apparatus and methods for the transfer of
therapeutic agents, such as therapeutic genes, to internal body
sites. The apparatus of the invention may be guided to diseased or
deficient organs, or other lesions, and deliver the therapeutic
agent in a targeted and controlled manner.
SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides a method of
delivering a negatively charged therapeutic agent to a target
location within a mammalian body. The method comprises the steps of
applying a multiplicity of alternating layers of at least one
cationic polyelectrolyte carrier and a multiplicity of layers of a
negatively charged therapeutic agent to at least one surface of an
insertable medical device. A washing step is employed between
application of the cationic polyelectrolyte and the negatively
charged therapeutic agent. The medical device is delivered to a
target site within the body, and upon reaching the target site the
negatively charged therapeutic agent is released into the target
site. The negatively charged therapeutic agent remains
qualitatively and quantitatively intact during the stages of
coating, washing, delivery and release.
[0009] In a preferred embodiment of this invention, the at least
one cationic polyelectrolyte carrier is human serum albumin,
gelatin, chitosan or a combination thereof.
[0010] In a more preferred embodiment of this invention, the outer
coating layer of the cationic polyelectrolyte carrier is chitosan,
gelatin or both, which cationic polyelectrolyte carriers affect the
time of release of the negatively charged therapeutic agent from
the insertable medical device upon delivery. The length of the time
lag could be controlled by the type and amount of the cationic
polyelectrolyte carrier used.
[0011] The device of this invention may compose a negatively
charged, neutral, or positively charged structure such as
polystyrene, polyethylene film, or glass. In a preferred embodiment
of this invention, a balloon catheter, having a balloon in a
diameter of about 0.4 cm, and a length of about 1.5, is used.
[0012] In yet another preferred embodiment of this invention, the
negatively charged therapeutic agent is a polynucleotide and in a
more preferred embodiment of this invention, the polynucleotide is
a naked DNA, DNA inserted into a viral or non-viral vectors.
[0013] Another preferred embodiment of this invention provides an
insertable medical device for insertion into a mammalian body,
wherein the insertable medical device has a multiplicity of
alternating layers of at least one cationic polyelectrolyte and a
biologically effective amount of a negatively charged therapeutic
agent, which are adsorbed on to a surface of the insertable medical
device. The amount of adsorbed negatively charged therapeutic agent
increases linearly with the number of the layers of same applied
and entrapped onto the surface of the medical device.
[0014] In a preferred embodiment of this invention, the insertable
medical device is, for example, a stent or a balloon catheter. In a
more preferred embodiment of this invention an outer coating of the
insertable medical device is employed to delay the release of the
negatively charged therapeutic agent. The outer coating is
preferably gelatin, and more preferably chitosan.
[0015] In another aspect of this invention, there is provided a
method for delivering a therapeutic agent that prevents or treats
angiogenesis, restenosis, cardiomyopathy, cystic fibrosis, or
malignant cell proliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a histogram showing the effect of pH on the amount
of DNA released from the surface of an insertable device. The
amount of released DNA from 10 layers of coating was measured at pH
3 and 4.
[0017] FIG. 2 is a graph showing the relationship between the
number of DNA layers and the amount of DNA adsorbed on the surface
of a medical device. Released DNA was measured against the number
of layers of DNA coatings on the surface of the medical device.
[0018] FIG. 3 is a photographic image of a DNA coated balloon
catheter (ethydium bromide stained) before and after the DNA
release. 1: DNA coated balloon catheter (stained with ethydium
bromide); 2: DNA coated balloon catheter after in vitro release
stained with ethydium 20 bromide; and 3: control uncoated balloon
catheter.
[0019] FIG. 4 is a graph showing release kinetics studies using
gelatin or chitosan coatings
[0020] .box-solid. without outer coating
[0021] .diamond. with gelatin coating (2%)
[0022] .quadrature. with chitosan coating (10 ppm)
[0023] .diamond. with chitosan coating (20 ppm)
[0024] with chitosan coating (40 ppm)
[0025] FIG. 5 is a histogram showing transfection rate of HEK 293
cells with DNA released from a balloon medical device. Columns 1-6
each represents the following:
[0026] 1: pRE-Luc+Lipofectamine; 2: pRE-Luc released from the
balloon+Lipofectamine;
[0027] 3: pRE-Luc; 4: DNA+Chitosan coated surface; 5: DNA+gelatin
coated surface; 6: DNA coated surface.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Definitions
[0029] As used herein, the following terms are defined as
follows:
[0030] "Therapeutic agent" as used herein includes any compounds or
compositions that induce a biological/medical reaction in vitro, in
situ, or in vivo settings.
[0031] "Negatively charged therapeutic agent" as used herein,
encompasses therapeutic agents that are negatively charged, either
naturally or synthetically. A negative charge may be added by any
known chemical means or biological means (i.e., addition or
deletion of functionalities, substitutions, or mutations).
[0032] "Therapeutic polynucleotide" as used herein includes nucleic
acids with and without carrier vectors, compacting agents, virus,
polymers, proteins, or targeting sequences.
[0033] "Stenosis" refers to a stricture of any bodily canal.
[0034] "Stent" refers to any tubular structure used to maintain or
support a bodily orifice or cavity.
[0035] "Balloon catheter" refers to a tubular instrument with a
balloon or multiple balloons that can be inflated or deflated
without removal after insertion into the body.
[0036] "Washing solution" according to this invention is water, any
suitable buffers or detergents, solvents or a combination
thereof.
[0037] "Surface" according to this invention means any portions of
any parts of an insertable medical device, or a combination of
different portions of different surfaces, of an insertable medical
device.
[0038] "Effective expression-inducing amount", as described herein,
means amount of a polynucleotide that effectuates expression of a
polypeptide encoded by a gene contained in such polynucleotide.
[0039] "Qualitatively and quantitatively intact", as described
herein, means substantially the same biological activity and
substantially the same amount. Substantially means at least about
90%.
DETAILED DESCRIPTION OF THE INVENTION
[0040] This invention describes a medical device and a method to
deliver a negatively charged therapeutic agent within the
vasculature of a patient. The negatively charged therapeutic agent
is adsorbed onto one or more sites or surfaces of a medical device,
thereby forming a coated surface, by a controlled adsorption
technique. When the coated surface(s) comes into contact with the
patient's blood, the negatively charged therapeutic agent is
released with a short controlled lag time of about 1 to several
minutes (for example, 1, 5 or 10 minutes) to allow the medical
device to reach the target site.
[0041] The method and medical device of this invention, as
described herein, maximize the amount of a negative therapeutic
agent that can be adsorbed to the medical device and control the
release of the negatively charged therapeutic agent, with only
minimal perturbation, at the target site. The method and medical
device, as described herein, use clinically acceptable
polyelectrolytes biopolymers such as human serum albumin (HSA) to
build the negatively charged therapeutic agent onto the surface of
medical device.
[0042] Washing is employed between application of each alternate
layers of one or more polyelectrolytes and the negatively charged
therapeutic agent. Washing ensures that the negatively charged
therapeutic agent is stably entrapped and not just precipitated on
the surface of the device. This method of coating the medical
device provides a more reproducible and controllable adsorption and
release kinetics of a negatively charged therapeutic agent
adsorption and release.
[0043] The medical device used in this invention is any insertable
medical device, including, for example, stents, catheters, or
balloon catheters. A preferred medical device for use with the
present invention is a balloon catheter. The medical device of this
invention can be used, for example, in any application for
treating, preventing, or otherwise affecting the course of a
disease or tissue or organ dysfunction. For example, the medical
instrument of the invention can be used to induce or inhibit
angiogenesis, or to prevent or treat restenosis, cardiomyopathy, or
other dysfunction of the heart, and is particularly applicable to
angioplasty treatment.
[0044] Additionally, the method and medical device described herein
can be used, for example, in treating cystic fibrosis or other
dysfunction of the lung, for treating or inhibiting malignant cell
proliferation, for treating any malignancy, and for inducing nerve,
blood vessel or tissue regeneration in a particular tissue or
organ.
[0045] Specific examples of the negatively charged therapeutic
agent used in conjunction with the present invention includes, for
example, any negatively charged compounds or compositions that are
negatively charged, either naturally or synthetically by means of
known chemical methods. In particular, the terms "therapeutic
agents" and "drugs" are used interchangeably herein and include
pharmaceutically active compounds and compositions, polynucleotides
with and without carrier vectors such as lipids, compacting agents
(such as histones), virus, polymers, proteins, and the like, with
or without targeting sequences.
[0046] Specific examples of the polynucleotide used in conjunction
with the present invention include, for example, oligonucleotides,
ribozymes, anti-sense oligonucleotides, DNA compacting agents,
gene/vector systems (i.e., any vehicle that allows for the uptake
and expression of nucleic acids), nucleic acids (including, for
example, recombinant nucleic acids; naked DNA, cDNA, RNA; genomic
DNA, cDNA or RNA in a non-infectious vector or in a viral vector
and which further may have attached peptide targeting sequences;
antisense nucleic acid (RNA or DNA); and DNA chimeras which include
gene sequences and encoding for ferry proteins such as membrane
trans locating sequences ("MTS") and herpes simplex virus-I
("VP22"), and constitutive housekeeping genes which are
theoretically expressed in all cell types.
[0047] Non-limiting examples of virus vectors or vectors derived
from viral sources include adenoviral vectors, herpes simplex
vectors, papilloma vectors, adeno-associated vectors, retroviral
vectors, and the like. The use of adenovirus is particularly
preferred.
[0048] Other examples of the therapeutic agent include any of the
following compounds and compositions, provided that they are made
to be negatively charged, using any known chemical and/or
biological method. Any of these modifications is routinely made by
one skilled in the art. These compounds include anti-thrombogenic
agents such as heparin, heparin derivatives, urokinase, and PPACK
(dextrophenylalanine proline arginine chloromethylketone);
antioxidants such as probucol and retinoic acid; angiogenic and
anti-angiogenic agents and factors; agents blocking smooth muscle
cell proliferation such as rapamycin, angiopeptin, and monoclonal
antibodies capable of blocking smooth muscle cell proliferation;
anti-inflammatory agents such as dexamethasone, prednisolone,
corticosterone, budesonide, estrogen, sulfasalazine, acetyl
salicylic acid, and mesalamine; calcium entry blockers such as
verapamil, diltiazem and nifedipine;
antineoplastic/antiproliferative/anti-mitotic agents such as
paclitaxel, 5-fluorouracil, methotrexate, doxorubicin,
daunorubicin, cyclosporine, cisplatin, vinblastine, vincristine,
epothilones, endostatin, angiostatin and thymidine kinase
inhibitors; antimicrobials such as triclosan, cephalosporins,
aminoglycosides, andnitorfurantoin; anesthetic agents such as
lidocaine, bupivacaine, and ropivacaine; nitric oxide (NO) donors.
such as lisidomine, molsidomine, L-arginine, NO-protein adducts,
NO-carbohydrate adducts, polymeric or oligomeric NO adducts;
anti-coagulants such as D-Phe-Pro-Arg chloromethyl ketone, an RGD
peptide-containing compound, heparin, antithrombin compounds,
platelet receptor antagonists, anti-thrombin antibodies,
anti-platelet receptor antibodies, enoxaparin, hirudin, Warafin
sodium, Dicumarol, aspirin, prostaglandin inhibitors, platelet
inhibitors and tick antiplatelet factors; vascular cell growth
promotors such as growth factors, growth factor receptor
antagonists, transcriptional activators, and translational
promotors; vascular cell growth inhibitors such as growth factor
inhibitors, growth factor receptor antagonists, transcriptional
repressors, translational repressors, replication inhibitors,
inhibitory antibodies, antibodies directed against growth factors,
bifunctional molecules consisting of a growth factor and a
cytotoxin, bifunctional molecules consisting of an antibody and a
cytotoxin; cholesterol-lowering agents; vasodilating-agents; agents
which interfere with endogeneus vascoactive mechanisms; survival
genes which protect against cell death, such as anti-apoptotic
Bcl-2 family factors and Akt kinase; and combinations thereof.
[0049] Polynucleotide sequences useful in practice of the invention
include DNA or RNA sequences having a therapeutic effect after
being taken up by a cell. Examples of therapeutic polynucleotides
include anti-sense DNA and RNA; DNA coding for an anti-sense RNA;
or DNA coding for tRNA or rRNA to replace defective or deficient
endogenous molecules. The polynucleotides of the invention can also
code for therapeutic proteins or polypeptides. A polypeptide is
understood to be any translation product of a polynucleotide
regardless of size, and whether glycosylated or not. Therapeutic
proteins and polypeptides include as a primary example, those
proteins or polypeptides that can compensate for defective or
deficient species in an animal, or those that act through toxic
effects to limit or remove harmful cells from the body.
[0050] In addition, the polypeptides or proteins, DNA of which can
be incorporated, include without limitation, angiogenic factors and
other molecules competent to induce angiogenesis, including acidic
and basic fibroblast growth factors, vascular endothelial growth
factor, hif-1, epidermal growth factor, transforming growth factor
.alpha. and .beta., platelet-derived endothelial growth factor,
platelet-derived growth factor, tumor necrosis factor .alpha.,
hepatocyte growth factor and insulin-like growth factor; growth
factors; cell cycle inhibitors including CDK inhibitors;
anti-restenosis agents, including p15, p16, p18, p19, p21, p27,
p53, p57, Rb, nFkB and E2F decoys, thymidine kinase ("TK") and
combinations thereof and other agents useful for interfering with
cell proliferation, including agents for treating malignancies; and
combinations thereof. Still other useful factors, which can be
provided as polypeptides or as DNA encoding these polypeptides,
include monocyte chemoattractant protein ("MCP-I"), and the family
of bone morphogenic proteins ("BMP's"). The known proteins include
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8,
BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16.
Currently preferred BMP's are any of BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6 and BMP-7. These dimeric proteins can be provided as
homodimers, heterodimers, or combinations thereof, alone or
together with other molecules. Alternatively or, in addition,
molecules capable of inducing an upstream or downstream effect of a
BMP can be provided. Such molecules include any of the "hedgehog"
proteins, or the DNA's encoding them.
[0051] The amount of polynucleotide adsorbed is an effective
expression-inducing amount. As used herein, the term "effective
expression-inducing amount" means that amount of the polynucleotide
that effectuates expression of a gene product encoded by such
polynucleotide. Means for determining an effective
expression-inducing amount of a polynucleotide are well known in
the art. For example, an effective expression-inducing amount of
the polypeptide of this invention is from about 0.3 to about 10
.mu.g/cm.sup.2/layer, preferably from about 0.5 to about 0.9
.mu.g/cm.sup.2/layer. The amount of polynucleotide adsorbed onto
the surface of the medical device is linearly proportional to the
number of layers applied thereto. At least up to 40 layers of a
therapeutic agent could be applied without affecting the properties
of the medical device. Preferably from about 4 to about 60 layers,
more preferably from about 10 to about 50 layers and most
preferably from about 20 about 40 layers of a therapeutic agent are
applied.
[0052] Polynucleotides, for example, naked DNA, DNA plus vector or
DNA delivery complex is captured on the surface of the medical
device by a cationic polyelectrolyte carrier. Viral or non-viral
vectors could be used to potentiate the transfection efficiency of
the released DNA. For example, a virus culture, such as adenovirus
could be layered on the surface of the insertable medical device.
The concentration of the virus solution significantly affects the
amount of the viral particles, which is incorporated into the
layers on the surface of the insertable medical device. The release
kinetics are reproducible and controlled. The released DNA is
bioactive with little decrease of potency.
[0053] Biological activity of the DNA released from the medical
device was studied by transfection of HEK 293 cells in vitro. The
results indicated that the biological activity of the released DNA
was the same as the control. Similar controlled-adsorption
techniques were used to adsorb adenoviruses on to the balloon
surface.
[0054] Any suitable surface of the medical instrument may be
coated. The surfaces to be coated may comprise any medically
acceptable material, such as, for example, carboxylated and
aminated polystyrene, and silanized glass.
[0055] As cationic polyelectrolyte carriers, any medically
acceptable polymers or copolymers, or natural polymers such as
human serum albumin, gelatin, chitosan and the like may be used.
The natural polymers are adsorbed onto a desired surface of the
medical device for coating. It is not necessary that an entire
surface is coated, rather, merely a portion of a surface may be
coated.
[0056] The coated medical device is inserted into the patient and
directed to the target site. When the coated surface comes into
contact with blood, the charge interaction of the cationic polymer
and the negatively charged therapeutic agent is disrupted due to a
charge screening effect and because the charge density of the
polymer is greatly decreased at physiological pH. In addition,
proteolytic degradation of the gelatin or HAS may also contribute
to the dissociation of the polymer-drug complex.
[0057] To delay dissociation of the adsorbed therapeutic agent when
a medical device coating with same is inserted into the blood
stream, a more cationic and more hydrophobic polymer layer can be
applied at the outer coating. The quantity and quality of the
biopolymer, particularly, the outer biopolymer, is directly
proportional to the duration of lag time achieved before the
release occurred. Chitosan, a natural polysaccharide derived from
crab shells, was used and shown to serve this purpose. Other
polymers can also be used to fine-tune the release kinetics of the
therapeutic agent from the coated surface. For example, with a thin
coating of condensed gelatin or chitosan, a short lag time of about
1-2 minutes is achieved before release occurs . Without the use of
gelatin or chitosan, 100% of the DNA is released at physiological
pH within minutes.
[0058] Organs and tissues that are treated by the methods of the
present invention include any mammalian tissue or organ, whether
injected in vivo or ex vivo. Non-limiting examples include
[0059] the heart, lung, brain, liver, skeletal muscle, smooth
muscle, kidney, bladder, intestines, stomach, pancreas, ovary,
prostate, cartilage and bone.
[0060] The negatively charged therapeutic agents, according to the
invention, can be used, for example, in any application for
treating, preventing, or otherwise affecting the course of a
disease or tissue or organ dysfunction. For example, the methods of
the invention can be used to induce or inhibit angiogenesis, as
desired, to prevent or treat restenosis, to treat a cardiomyopathy
or other dysfunction of the heart, for treating cystic fibrosis or
other dysfunction of the lung, for treating or inhibiting malignant
cell proliferation, for treating any malignancy, and for inducing
nerve, blood vessel or tissue regeneration in a particular tissue
or organ. Particularly, the negatively charged therapeutic agents
of this invention are used preferably in angioplasty. Having now
fully described the invention, the same would be more readily
understood by reference to specific examples which are provided by
way of illustration, and not intended to be limiting of the
invention, unless herein specified.
EXAMPLE 1
Effect of Surfaces and Polyelectrolytes on the DNA Release
[0061] Multilayered films of DNA were built up on various
negatively charged, neutral, and positively charged surfaces, by
spraying or dipping. The DNA adsorbed by HSA or gelatin was
released quickly whereas, due to the hydrophobicity of chitosan at
neutral pH, the DNA adsorbed by chitosan was released very slowly.
The result of this experiment is tabulated in Table 1 below. Table
1 shows natural polymers, as polyelectrolytes, are coated onto
several surfaces, which surfaces were modified by different
substrates. When different surfaces were dipped into a slightly
acidic solution containing a polynucleotide, the positively charged
coated surface induced adsorption of the polynucleotide (i.e.,
adsorption was driven by the charged interaction). Successive
layering of the surface with polyelectrolyte and DNA can be
repeated as many times as needed to maximize the amount of DNA
adsorbed to the surface. The alternate layers of polyelectrolyte
and polynucleotide are stable in a solution that is slightly acidic
and of low ionic strength.
1TABLE 1 The amount of DNA release with different polyelectrolyte
combinations and substrates Number Amount of DNA released Substrate
Formulation of layers (.mu.g/cm.sup.2) PEG/gelatin- Gelatin/DNA 4
1.23 (overnight) modified Glass Carboxylated PS HSA/DNA 4 1.24 (0.5
hr) Polyethylene film HSA/DNA 4 1.14 (0.5 hr) PET balloon HSA/DNA 4
1.29 (0.5 hr) PET balloon HSA/DNA 10 3.28 (overnight) PET balloon
Gelatin/DNA 10 2.62/5.13 (0.5 hr/4 d) PET balloon Chitosan/DNA 20
0/0.86 (1 hr/17 hrs)
EXAMPLE 2
Effects of pH on the Amount of DNA Released
[0062] The effect of pH on the amount of DNA adsorbed was
investigated by alternating adsorption of DNA and HSA at different
pHs. The results, as shown in FIG. 1, indicated that HSA adsorption
is optimal at pH 4.0; no DNA could be adsorbed at pH 5.0 or higher.
The relationship between the number of DNA layers and adsorbed
amount of DNA was investigated by alternating adsorption of DNA and
HSA at pH4.0. The results, as demonstrated in FIG. 2, showed that
the amount of DNA absorbed increased linearly with the number of
DNA layers on the surface of the medical device.
EXAMPLE 3
The Release Kinetics of the Adsorbed DNA from the Surface of the
Medical Device
[0063] Release kinetics studies indicated that the adsorbed
polynucleotide could be released completely within 5 minutes. (See
FIG. 3 which shows that the adsorbed DNA was released almost
completely from the surface of a coated balloon catheter.) When the
medical device is coated with a condensed gelatin coating, the
release rate of the adsorbed DNA was reduced slightly, whereas when
the device was coated with a thin layer of chitosan, the release
rate of the adsorbed DNA was decreased remarkably. The release
kinetics of the adsorbed DNA, as shown in FIG. 4, was also shown to
be dependant on the thickness of chitosan coating (i.e., the
concentration of chitosan solution when the dipping time was
fixed).
EXAMPLE 4
Biological Activity of the Released DNA
[0064] The biological activity of DNA, released from the surface of
a coated medical device, was investigated by transfecting HEK 293
cells in vitro. The result of this study, as shown in FIG. 5,
indicates that the released DNA was still biologically active. A
comparison between columns 1 and 2 of FIG. 5 shows that the DNA
released from the medical device coated with gelatin or chitosan,
similar to the naked DNA, had a high transfection efficiency.
Occasionally, cationic gelatin complexes with DNA in the soluble
form and transfects cells in culture better than naked DNA.
EXAMPLE 5
Feasibility of Delivering Adenovirus
[0065] Using similar adsorption technique with gelatin as the
polycation, .sup.125I labeled recombinant adenovirus, encoding the
Lac Z gene, was adsorbed onto a balloon surface. The result of this
experiment is shown in Table 2 below. Table 2 indicates that the
amount of plaque forming units and virus particles of adenovirus,
released or remained on the balloon after release delivery,
constitutes a major portion of the total amount of virus found on
the surface of the balloon.
2TABLE 2 Feasibility of delivering adenovirus Total amount of virus
Amount of adenovirus Readings (pfu) (particles) (pfu/cm.sup.2)
(vp/cm.sup.2) Calibration Standard 538.5 .+-. 52 6.4 .+-. 0.6
.times. 10.sup.7 1.6 .+-. 0.2 .times. 10.sup.9 -- -- Released
adenovirus 47 .+-. 9.5 5.6 .+-. 1.1 .times. 10.sup.6 1.4 .+-. 0.3
.times. 10.sup.8 4.0 .+-. 0.8 .times. 10.sup.6 1.0 .+-. 0.2 .times.
10.sup.8 Virus remained 168.5 .+-. 58 2.0 .+-. 0.7 .times. 10.sup.7
5.0 .+-. 1.7 .times. 10.sup.8 1.4 .+-. 0.5 .times. 10.sup.7 3.5
.+-. 1.2 .times. 10.sup.8 balloon after release
[0066] Table 2 shows the amount of adenovirus released in 60
minutes in 10% serum culture media from a 10-layered balloon.
EXAMPLE 6
DNA Delivery via Negatively Charged Polystyrene (PS) Surface
[0067] Carboxylated polystyrene (PS) wells were treated with 360
.mu.l (per well) of 0.1% human serum albumin (HSA) in 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) at room temperature
(RT) overnight. The wells were washed thoroughly with water and
then treated with 360 .mu.L (per well) of DNA (247 .mu.g/ml) in 25
mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) at RT for 0.5
hr. The wells were washed once with 360 .mu.l (per well) of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) and then treated
again with 360 .mu.l of 0.1% HSA in 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) at RT for 0.5 hr. The wells were
washed once with 360 .mu.l (per well) of 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) and then treated again with 360
.mu.l (per well) of DNA (247 ug/ml) in 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) at RT for 0.5 hr. The preceding
washing steps were repeated until multilayer of DNA layers were
adsorbed. The wells were washed once with 360 .mu.l (per well) of
25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) and then
treated with 360 .mu.l of 1.times. phosphate buffered saline (PBS)
at RT for 0.5 hr .The amount of DNA released in PBS was determined,
and shown in Table 3 below.
3TABLE 3 Relationship between the number of DNA layers and the
amount of DNA Released A B C D Number of DNA layers 1 2 3 4
Released DNA (.mu.g/cm.sup.2) 0.105 0.493 0.806 1.240
EXAMPLE 7
DNA Delivery via Positively Charged Glass Surface
[0068] Two pieces of polyethylene glycol (PEG)/gelatin-modified
glass plates were treated with 2 ml of DNA (140 .mu.g/ml) in 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 5.5) at 37.degree. C.
for 1 hr. The plates were washed once with 3 ml of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) and then treated
with 2 ml of 0.05% gelatin (A, 175) in 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.8) at 37.degree. C. for 1 hr. The
plates were washed once with 3 ml of 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) and then treated again with 2 ml
of DNA (88 .mu.g/ml) in 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4
buffer (pH 5.5) at 37.degree. C. for 1 hr. The preceding washing
steps were repeated until several layers of DNA layers were
adsorbed. The plates were washed once with 3 ml of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) and then dipped in
2 ml of 1.times.PBS at RT for 1 hr. The amount of DNA released in
PBS is shown in Table 4 below.
4TABLE 4 DNA release from glass surface. A B Number of DNA layers 4
9 Released DNA (.mu.g/cm.sup.2) 1.231 3.923
EXAMPLE 8
cDNA Delivery via Neutral PET Balloon Surface
[0069] Two balloons (each having the diameter of 0.4 cm, length of
1.5 cm, and a surface area of ca. 1.88 cm.sup.2) were treated with
5 ml of 0.1% HSA in 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer
(pH 4.0) at RT for 2 hours. The balloons were washed thoroughly
with water and then treated with 5 ml of DNA (141 .mu.g/ml, p 43
hGFP) in 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) at
RT for 0.5 hr. The balloons were washed once with 5 ml of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0) and then treated
again with 5 ml of 0.1% HSA in 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) at RT for 0.5 hr. Balloons were
washed once with 5 ml of 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4
buffer (pH 4.0) and then treated again with 5 ml of DNA (141
.mu.g/ml) in 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 4.0)
at RT for 0.5 hr. The preceding washing steps were repeated until 4
DNA layers were adsorbed. Results are tabulated in Table 5
below.
5TABLE 5 DNA delivery on two balloons. A B Readings (ng/ml) 55 54
Released DNA (.mu.g) 2.42 2.376 Released DNA (.mu.g/cm.sup.2) 1.287
1.264 A: Balloon was washed once with 5 ml of 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 4.0) and then dipped in 4 ml of I
.times. PBS at RT for 0.5 hr. B: Balloon was not washed and
directly dipped in 4 ml of 1 .times. PBS at RT for 0.5 hr. The
amount of DNA released in PBS was determined (200 .mu.l was taken
into 2 ml of test solution).
EXAMPLE 9
Transfection of HEK 293 Cells in Vitro
[0070] In a twelve-well tissue culture plate, 8.times.10.sup.4 HEK
293 cells per well were seeded in 1 ml of the appropriate complete
growth medium (10% serum) and incubated at 37.degree. C. in a
CO.sub.2 incubator for 1 day. The culture medium was then removed
and the transfection medium was added to the cells. The cells were
then divided into 6 different groups:
[0071] Group 1: 2 .mu.g of DNA (Luci) and 2 .mu.l of lipofectamine
in 1 ml of serum-free medium; Group 2: Released DNA (Luci, 20
layers) and 2 .mu.l of lipofectamine in 1 ml of serum-free medium.
Group 3: 2 .mu.g of DNA (Luci) in I ml of serum-free medium. Group
4: Released DNA (Luci, 20 layers) with an outermost chitosan
coating. Group 5: Released DNA (Luci, 20 layers) with an outermost
gelatin coating. Group 6: Released DNA (Luci, 20 layers). The cells
were incubated at 37.degree. C. in a CO.sub.2 incubator for three
days. The media was removed from the cells and the cells were
rinsed once with 1.times.PBS. Cell Culture Lysis (200 .mu.l) was
added at the concentration of 1.times. Reagent per well to cover
the cells. The cells were incubated at room temperature for 10-15
minutes. The cell extract (about 20 .mu.l) was added to a
luminometer cuvette at room temperature, followed by 100 .mu.l of
Luciferase Assay Reagent, again at room temperature. The cuvette
was placed in the luminometer. Light emission was measured for ten
seconds. Protein concentration was determined using the Bio-Rad
protein assay kit. The results, as shown in FIG. 5, were expressed
as relative light units/min mg protein.
EXAMPLE 10
Adenovirus Adsorption
[0072] Two balloons [1.5 cm (L).times.0.3 cm (D), 1.41 cm.sup.2]
were treated with 1 ml of 0.1% gelatin (A, 300) in 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 5.0) at RT for 2 hours.
The balloons were washed three times with 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 5.0) and then treated with 1 ml of
adenovirus (1.6.times.10.sup.9 pfu/ml or 4.times.10 vp/ml
containing a small amount of .sup.125I -labeled adenovirus) in 25
mM HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 5.0) at RT for 2
minutes. The balloons were washed once with 1 ml of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 5.0) and then treated
with 1 ml of 0.1% gelatin (A, 300) in 25 mM HAc-NaAc/25 mM
Na.sub.2SO.sub.4 buffer (pH 5.0) at RT for 2 minutes. The balloons
were washed once with 1 ml of 25 mM HAc-NaAc/25 mM Na.sub.2SO.sub.4
buffer (pH 5.0) and then treated again with 1 ml of adenovirus
(1.6.times.10.sup.9 pfu/ml or 4.times.10.sup.10 vp/ml) containing a
small amount of .sup.125I-labeled adenovirus in 25 mM HAc-NaAc/25
mM Na.sub.2SO.sub.4 buffer (pH 5.0) at RT for 2 minutes. The
preceding two steps were repeated until ten adenovirus layers were
adsorbed. The balloons were washed once with 1 ml of 25 mM
HAc-NaAc/25 mM Na.sub.2SO.sub.4 buffer (pH 5.0) and dipped in 1 ml
of 10% serum culture medium for 60 min. The amount of adenovirus
was determined by comparing the amount of radioactivity of
.sup.125I (10 ml of counting medium was used for each sample, Count
1 minute). The result of this experiment is shown in Table 5,
above.
EXAMPLE 11
In Vivo Delivery of DNA to Heart
[0073] To stimulate angiogenesis or collateral blood flow in the
adult rat heart, a balloon catheter, is coated with 40 layers of a
DNA encoding human fibroblast growth factor-5 (hFGF-5) and is
inserted into a blood vessel that perfuses the heart. Rats have
been sacrificed at 3 weeks following injection and capillary
density was measured by computerized light microscopy. The results
have shown that a direct injection of a fibroblast growth factor -5
expression vector stimulates collateral vessel formation in areas
of injected myocardium.
* * * * *