U.S. patent application number 11/497482 was filed with the patent office on 2006-11-30 for antisense restenosis composition and method.
This patent application is currently assigned to AVI Biopharma, Inc.. Invention is credited to Patrick L. Iversen, Dwight D. Weller.
Application Number | 20060269587 11/497482 |
Document ID | / |
Family ID | 36821690 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269587 |
Kind Code |
A1 |
Iversen; Patrick L. ; et
al. |
November 30, 2006 |
Antisense restenosis composition and method
Abstract
The present invention provides an improved method for reducing
the risk or severity of restenosis following cardiac angioplasty.
The method includes administering to a target vessel region, a
morpholino antisense compound having uncharged
phosphorus-containing backbone linkages, and spanning the start
codon of a human c-myc mRNA. Also disclosed are novel antisense
compounds and compositions, and a method for assaying the
effectiveness of antisense delivery and uptake to a target vessel
region.
Inventors: |
Iversen; Patrick L.;
(Corvallis, OR) ; Weller; Dwight D.; (Corvallis,
OR) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
AVI Biopharma, Inc.
|
Family ID: |
36821690 |
Appl. No.: |
11/497482 |
Filed: |
July 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09493427 |
Jan 29, 2000 |
7094765 |
|
|
11497482 |
Jul 31, 2006 |
|
|
|
60117846 |
Jan 29, 1999 |
|
|
|
Current U.S.
Class: |
424/426 ;
514/44A; 514/81; 544/81 |
Current CPC
Class: |
A61K 31/07 20130101 |
Class at
Publication: |
424/426 ;
514/044; 514/081; 544/081 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 31/675 20060101 A61K031/675; C07F 9/6533 20060101
C07F009/6533 |
Claims
1. A method for treating a vascular injury site in a human patient
by reducing restenosis at the site, said method comprising
administering to the patient, by intravascular delivery, an
antisense compound having: (i) morpholino subunits linked together
by phosphorodiamidate linkages, 2 atoms long, joining the
morpholino nitrogen of one subunit to the 5' exocyclic carbon of an
adjacent subunit, and (ii) a sequence of bases attached to the
subunits and containing the sequence identified by SEQ ID NO:1.
2. The method of claim 1 wherein the antisense compound has
intersubunit linkages of the form: ##STR1## where X is an
unsubstituted, monosubstituted or disubstituted nitrogen.
3. The method of claim 1, wherein the antisense compound has from 8
to 40 nucleotides, including a targeting sequence complementary to
a region spanning the start codon of human c-myc mRNA, where the
nucleotide sequence is SEQ ID NO:1.
4. The method of claim 3, wherein the mode of administration is
selected from the group consisting of: (a) contacting the region of
the vessel with a reservoir containing the antisense compound, and
introducing the compound from the reservoir into the vessel by
iontophoresis or electroporation; (b) injecting the compound from
the catheter directly into the region of the vessel, under
pressure, through injectors contained on the surface of the
catheter balloon, where said injectors are capable of penetrating
the tunica media in the vessel; (c) injecting into or contacting
the region of the vessel, microparticles containing the antisense
compound in entrapped form; (d) contacting the region of the vessel
with a hydrogel coating contained on the surface of the catheter
balloon, and containing the antisense compound is diffusible form;
and (e) contacting the region of the vessel with a stent having an
outer surface layer containing the antisense compound in diffusible
form.
5. The method of claim 4, wherein the amount of antisense compound
administered is between 0.5 and 20 mg.
6. The method of claim 4, in the mode of administration set forth
in (a), wherein the antisense compound is contained in a volume
between two inflated balloons in the catheter, the compound
contains a net charge, and the volume is subjected to pulsed
electric fields effective to iontophoretically drive the compound
into the vessel region.
7. The method of claim 4, in the mode of administration set forth
in (a), wherein the antisense compound is contained in a volume
between two inflated balloons in the catheter, and the volume is
subjected to pulsed electric fields effective to facilitate
compound uptake into vessel-region cells by electroporation.
8. The method of claim 4, in the mode of administration set forth
in (b), wherein the catheter balloon has a plurality of
outer-facing channels that communicate with a distal-tip reservoir,
each channel having one or more injection ports, and said injecting
includes forcing a solution or suspension of the antisense compound
from said reservoir through said injection ports when the balloon
is in an inflated position.
9. The method of claim 4, in the mode of administration set forth
in (c), wherein the catheter has a distal end reservoir, the
microparticles are contained as a particle suspension in the
reservoir, and said injecting includes forcing the suspension out
of the catheter through a catheter surface in contact with the
vessel region.
10. The method of claim 4, wherein the particles are microbubbles
containing the antisense compound in entrapped form.
11. The method of claim 4, in the mode of administration set forth
in (e), wherein the stent is biodegradable.
12. The method of claim 4, wherein the compound is derivatized with
triethyleneglycol attached to the 5' end of the compound, and the
compound is administered from a solution containing at least 30
mg/ml of the antisense compound.
13. A morpholino antisense compound having: (i) morpholino subunits
linked together by phosphorodiamidate linkages, 2 atoms long,
joining the morpholino nitrogen of one subunit to the 5' exocyclic
carbon of an adjacent subunit, and (ii) a sequence of bases
attached to the subunits and containing the sequence identified by
SEQ ID NO:1, for the preparation of a pharmaceutical composition
for use in inhibiting restenosis in a patient.
14. A morpholino antisense compound having (i) from 8 to 40
nucleotides, including a targeting nucleic acid sequence
complementary to a region that spans the start codon of a human
c-myc mRNA gene, and (ii) phosphorous-containing intersubunit
linkages.
15. The compound of claim 13 having intersubunit linkages of the
form: ##STR2## where X is an unsubstituted, monosubstituted or
disubstituted nitrogen.
16. The compound of claim 13, wherein the compound is derivatized
with triethyleneglycol attached to the 5' end of the compound.
17. The compound of claim 13, which is entrapped in liposomal or
biodegradable microparticles.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/493,427, filed Jan. 29, 2000, which claims
priority to U.S. Provisional Application No. 60/117,846, filed Jan.
29, 1999, all of which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for treating restenosis, and in particular to an antisense
composition directed against c-myc, and a method of administering
the composition to reduce the risk of restenosis in transluminal
angioplasty, such as percutaneous transluminal coronary angioplasty
(PTCA).
REFERENCES
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et al., Circulation, 90(5):2474-80, (1994). [0031] Oberhoff M; et
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BACKGROUND OF THE INVENTION
[0044] Transluminal coronary angioplasty was introduced in the late
1970's as a nonsurgical treatment for obstructive coronary artery
disease. Since its introduction, major advances in equipment and
techniques have led to widespread use of the method for treating
coronary artery disease and angina. Typically, the procedure
involves placing a balloon-tip catheter at the site of occlusion,
and disrupting and expanding the occluded vessel by inflating the
catheter balloon.
[0045] Despite improvements in equipment and techniques, restenosis
persists as the limiting factor in the maintenance of vessel
patency in angioplasty, occurring in 30% to 50% of patients, and
accounting for significant morbidity and health care expenditures
(Casterella). Post-angioplasty restenosis is a segmentally limited,
wound healing response to a traumatization of the vascular wall.
Studies with animal models and human autopsy plaque tissue indicate
a cascade-like course of events triggered by (a) destruction of
endothelial and subendothelial structures, (b) traumatization of
medial regions with rupture of the internal elastic lamina, (c)
release of thrombogenic factors such as collagen or tissue factor,
(d) stretching of smooth muscle cells with subsequent expression of
proto-oncogenes (c-fos, c-myc, c-myb), (e) release of growth
factors from cells of the bloodstream, endothelial cells and SMCs,
and (f) thrombin production with autocatalytic activation of the
SMC thrombin receptor (Bauriedel).
[0046] Overlapping the inflammation period, granulation begins 3
days after angioplasty. Proteinases such as plasmin as well as
collagenases induce the disintegration of extracellular matrix
structures, thereby modulating plaque formation, and lead to an
organelle-rich SMC phenotype within the intima and media.
Overlapping with the granulation period, induction of different
components of the extracellular matrix occurs 1-2 weeks after
angioplasty, possibly mediated by TGF-beta (phase of matrix
formation). Smooth muscle cells produce and secrete matrix proteins
such as tenascin, fibronectin, collagens and proteoglycans, and
thereby induce a marked increase of the neointimal plaque volume
(Bauriedel).
[0047] Clinical trials in restenosis prevention using various
revascularization devices, antiplatelet drugs, antithrombotic
drugs, and anti-inflammatory agents have produced limited
improvement in the incidence of restenosis. Also reported are
attempts to improve the risk or severity of restenosis with
intravascular stents (Savage, Eisenhower, Rubarteli, Gottman),
radiation therapy (Koh), and administration of anti-proliferative
drugs at the vessel injury site. The latter approach typically
employs the balloon catheter for introducing the therapeutic agent
at the vessel occlusion site (Dick, Roy, Dev, Kimura, Alfke,
Robinson 1997a, Robinson 1997b, Barath, Herdeg, Pavlides, Oberhoff,
Hodgkin, Hong, Consigny, Meyer, Fernadez-Ortiz, Lambert, and
Wilensky), or releasing drug from a stent (Teomin, Bartonelli,
Raman, Gibson).
[0048] Despite these advances, the incidence of restenosis, and the
inability to predict the response to treatment, remains a serious
risk factor in vascular angioplasty. It would therefore be
desirable to (i) provide a treatment method which shows efficacy in
reducing the incidence and severity of restenosis following
vascular angioplasty, (ii) is well tolerated by the patients, with
few or any side effects, and (iii) can be carried out with a
variety of therapeutic delivery methods.
[0049] It would also be desirable to provide improved therapeutic
compounds and compositions for carrying out the method, and a
simple, rapid clinical assay for monitoring effectiveness of the
delivery of a therapeutic compound to the vessel target site.
SUMMARY OF THE INVENTION
[0050] In one aspect, the invention includes a method of reducing
the risk of restenosis in a region of a patient's coronary vessel
which has been treated by coronary angioplasty using a catheter
with a distal-end expandable balloon, or which is at a vessel
junction formed in a coronary bypass operation. The method includes
administering to the patient, by direct local administration to the
vessel site or injury, a morpholino antisense compound having (i)
from 8 to 40 nucleotides, including a targeting base sequence that
is complementary to a region that spans the translational start
codon of a c-myc mRNA, and (ii) uncharged, phosphorous-containing
intersubunit linkages, in an amount effective to reduce the risk or
severity of restenosis in the patient.
[0051] The administering is carried out by (a) contacting the
region of the vessel with a reservoir containing the antisense
compound, and introducing the compound from the reservoir into the
vessel by iontophoresis or electroporation; (b) injecting the
compound from the catheter directly into the region of the vessel,
under pressure, through injectors contained on the surface of the
catheter balloon, where the injectors are capable of penetrating
the tunica media in the vessel; (c) injecting into or placing at
the region of the vessel, microparticles containing the antisense
compound in entrapped form; (d) contacting the region of the vessel
with a hydrogel coating contained on the surface of the catheter
balloon, and containing the antisense compound in diffusable form;
or (e) contacting the region of the vessel with a stent having an
outer surface layer containing the antisense compound in diffusable
form.
[0052] The antisense compound preferably has intersubunit linkages
selected from the group consisting of the structures presented in
FIGS. 2A-A-2D-D, and exemplified particularly by the
phosphorodiamidate linkage represented at FIG. 2B-B, where
X=NH.sub.2, Y=O, and Z=O. An exemplary sequence is the one
identified by SEQ ID NO:1.
[0053] For use in mode of administration (a), the antisense
compound is preferably contained in a volume between two inflated
balloons in the catheter, and the volume is subjected to pulsed
electric fields effective to ionotophoretically drive the compound
into region of the vessel.
[0054] For use in mode of administration (b), the catheter balloon
preferably has a plurality of outer-facing channels that
communicate with a distal-tip reservoir, where each channel having
one or more injection ports or fingers, and the injecting step
includes forcing a solution or suspension of the antisense compound
from the reservoir through the injection ports when the balloon is
in an inflated position.
[0055] For use in mode of administration (c), the catheter
preferably has a distal end reservoir, the microparticles are
contained as a particle suspension in the reservoir, and the
injecting step includes forcing the suspension out of the catheter
through a catheter surface in contact with the vessel region.
Exemplary particles include biodegradable polymer particles or
liposomes with entrapped antisense compounds or microbubbles
designed to release entrapped compound when subjected to ultrasonic
energy.
[0056] For use in mode of mode of administration (d), the hydrogel
coating is preferably designed to release the majority of the
antisense compound in the coating over a period of 5-60 minutes
following balloon angioplasty.
[0057] For use in mode (e), the stent may be biodegradable, and
designed to release the majority of the antisense compound in the
coating over a period of 5-60 minutes following balloon
angioplasty.
[0058] In a related aspect, the invention includes a method of
reducing the risk of restenosis in a region of a patient's coronary
vessel that has been treated by coronary angioplasty using a
catheter with a distal-end expandable balloon. The method includes
administering to the patient, by direct administration to the site
of injury, a morpholino antisense compound having (i) the base
sequence identified as SEQ ID NO:1, and (ii) a phosphorodiamidate
backbone shown in. FIG. 2B-B, where X=NH.sub.2, Y=O, and Z=O. The
antisense compound may be derivatized, e.g., at its 5' end, with a
moiety that enhances the solubility of the compound in aqueous
medium, and/or with a moiety that imparts a charge to the compound
at physiological pH. The compound is preferably delivered by direct
application of the compound to the target vessel region,
immediately following balloon angioplasty, or during a coronary
bypass operation, in an amount of between about 1-30 mg, to achieve
a final amount of compound administered to the target region of
between about 0.5 to 2 mg.
[0059] In another aspect, the invention includes a morpholino
antisense compound having (i) from 8 to 40 nucleotides, including a
targeting nucleic acid sequence complementary to a region that
spans the start codon of a human c-myc mRNA gene, and (ii)
uncharged, phosphorous-containing intersubunit linkages. The
intersubunit linkages are preferably selected from the group
consisting of the structures presented in FIGS. 2A-A-2D-D, as
exemplified particularly by the phosphorodiamidate linkage
represented at FIG. 2B-B, where X=NH.sub.2, Y=O, and Z=O. An
exemplified sequence is given by the sequence identified by SEQ ID
NO:1. The antisense compound may be derivatized, e.g., at its 5'
end, with a moiety that enhances the solubility of the compound in
aqueous medium, and/or with a moiety that imparts a charge to the
compound at physiological pH. The compound may be included in a
liposomal or other microparticle vehicle.
[0060] In still another aspect, the invention includes a method for
assaying the ability of an antisense compound to reach and interact
with c-myc mRNA in vessel cells, in a treatment method using
antisense compound to reduce the risk of restenosis. The method
includes (a) administering to the patient, a morpholino antisense
compound having a substantially uncharged backbone, and a sequence
that spans the start codon of a human c-myc mRNA, (b) at a selected
time after the compound is administered, taking a sample of a body
fluid from the subject, and (c) detecting in the sample, the
presence of a nuclease-resistant heteroduplex composed of the
antisense compound and the target RNA region.
[0061] These and other objects and features of the present
invention will become more fully apparent when the following
detailed description of the invention is read in conjunction with
the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1 shows several preferred subunits having 5-atom (A),
six-atom (B) and seven-atom (C-D) linking groups suitable for
forming polymers.
[0063] FIGS. 2A-A to 2D-D show the repeating subunit segment of
exemplary morpholino oligonucleotides, designated A-A through D-D,
constructed using subunits A-D, respectively, of FIG. 1.
[0064] FIG. 3 is a kinetic representation of the disappearance of
PMO monomer and appearance of RNA:PMO heterodimer in the plasma of
rats administered the P450 antisense PMO.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0065] The terms below, as used herein, have the following
meanings, unless indicated otherwise:
[0066] "Antisense" refers to an oligomer having a sequence of
nucleotide bases and a subunit-to-subunit backbone that allows the
antisense oligomer to hybridize to a target sequence in an RNA by
Watson-Crick base pairing, to form an RNA:oligomer heteroduplex
within the target sequence, typically with an mRNA. The oligomer
may have exact sequence complementarity to the target sequence or
near complementarity. These antisense oligomers may block or
inhibit translation of the mRNA, and/or modify the processing of an
mRNA to produce a splice variant of the mRNA. Studies conducted in
support of the present invention, for example, show that the
antisense compound represented by SEQ ID NO:1 interferes with c-myc
mRNA processing, leading to a truncated mRNA in which the normal
start codon and adjacent region has been spliced out of the
mRNA.
[0067] As used herein, the terms "compound", "agent", "oligomer"
and "oligonucleotide" may be used interchangeably with respect to
the antisense oligonucleotides of the invention. As used herein, a
"morpholino oligomer" refers to a polymeric molecule having a
backbone which supports bases capable of hydrogen bonding to
typical polynucleotides, wherein the polymer lacks a pentose sugar
backbone moiety, and more specifically a ribose backbone linked by
phosphodiester bonds which is typical of nucleotides and
nucleosides, but instead contains a ring nitrogen with coupling
through the ring nitrogen. A preferred "morpholino" oligonucleotide
is composed of morpholino subunit structures of the form shown in
FIG. 2B-B, where (i) the structures are linked together by
phosphorous-containing linkages, one to three atoms long, joining
the morpholino nitrogen of one subunit to the 5' exocyclic carbon
of an adjacent subunit, and (ii) P.sub.i and P.sub.j are purine or
pyrimidine base-pairing moieties effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide.
Exemplary structures for antisense oligonucleotides for use in the
invention include the morpholino subunit types shown in FIGS. 1A-D,
with the linkages shown in FIGS. 2A-A to 2D-D.
[0068] As used herein, a "nuclease-resistant" oligomeric molecule
(oligomer) is one whose backbone is not susceptible to nuclease
cleavage of a phosphodiester bond.
[0069] As used herein, an oligonucleotide or antisense oligomer
"specifically hybridizes" to a target polynucleotide if the
oligomer hybridizes to the target under physiological conditions,
with a Tm substantially greater than 37.degree. C., preferably at
least 50.degree. C., and typically 60.degree. C.-80.degree. C. or
higher. Such hybridization preferably corresponds to stringent
hybridization conditions, selected to be about 10.degree. C., and
preferably about 50.degree. C. lower than the thermal melting point
(T[m]) for the specific sequence at a defined ionic strength and
pH. At a given ionic strength and pH, the T[m] is the temperature
at which 50% of a target sequence hybridizes to a complementary
polynucleotide.
[0070] Polynucleotides are described as "complementary" to one
another when hybridization occurs in an antiparallel configuration
between two single-stranded polynucleotides. A double-stranded
polynucleotide can be "complementary" to another polynucleotide, if
hybridization can occur between one of the strands of the first
polynucleotide and the second. Complementarity (the degree that one
polynucleotide is complementary with another) is quantifiable in
terms of the proportion of bases in opposing strands that are
expected to form hydrogen bonds with each other, according to
generally accepted base-pairing rules.
[0071] As used herein, the term "c-myc antisense compound" refers
to a nuclease-resistant antisense morpholino compound having high
affinity (i.e., "specifically hybridizes") to a complementary or
near-complementary c-myc nucleic acid sequence, e.g., the sequence
including and spanning the normal AUG start site.
[0072] As used herein the term "analog" in reference to an oligomer
means a substance possessing both structural and chemical
properties similar to those of the reference oligomer.
[0073] As used herein, "effective amount" relative to an antisense
oligomer refers to the amount of antisense oligomer administered to
a mammalian subject, either as a single dose or as part of a series
of doses, that is effective to reduce the risk (incidence) or
severity (amount of occlusion) of restenosis, following balloon
angioplasty.
[0074] As used herein, the term "body fluid" encompasses a variety
of sample types obtained from a subject including, urine, saliva,
plasma, blood, spinal fluid, and other liquid sample of biological
origin, and may refer include cells or cell fragments suspended
therein, or the liquid medium and its solutes.
II. Compound and Composition
[0075] A. c-myc Antisense Compound
[0076] c-myc is a proto-oncogene which regulates cell growth and
differentiation, is involved in the process of vascular remodeling,
regulating smooth muscle cell proliferation and extracellular
matrix synthesis, in addition to playing a role in apoptosis.
Aberrant expression of c-myc is frequently observed in human
cancer. Aberrant, constitutive or overexpression of c-myc has been
associated with a number of human cancers including lung cancer,
colorectal cancer, breast cancer, bladder cancer, leukemia, lung
cancer, etc.
[0077] Several in vitro studies have demonstrated that
phosphorothioate oligodeoxynucleotides targeted against genes
involved in smooth muscle cell proliferation inhibit both
proliferation and migration. In one study in vivo administration of
phosphorothioate oligonucleotides targeted against c-myc using a
porous balloon catheter in a porcine coronary artery model (Shi),
and in another study phosphorothioate oligonucleotides delivered
intraluminally and targeted against c-myb, c-myc, cdc2 kinase, cdk2
kinase and proliferating cell nuclear antigen (PCNA) inhibited
neointimal formation after balloon injury in both the rat carotid
and porcine coronary artery models (Lee).
[0078] However, a similar study single endoluminal transcatheter
delivery of antisense oligonucleotides directed against cell cycle
regulatory proteins using a porous balloon catheter did not affect
neointima formation or vessel size (Robinson). The results of a
further study using phosphorothioate oligonucleotides directed
toward c-myb and c-myc indicated inhibition of smooth muscle cell
proliferation. However, the observed inhibition was clearly not via
an antisense mechanism, but was correlated with the presence of
four contiguous guanosine residues in the oligonucleotide sequence
in vitro in primary cultures of smooth muscle cells and in arteries
ex vivo (Burgess).
[0079] In accordance with the present invention, it has been
discovered that a morpholino antisense compound having (i) from 8
to 40 nucleotides, including a targeting base sequence that is
complementary to a region that spans the translational start codon
of a c-myc mRNA and (ii) uncharged, phosphorous-containing
intersubunit linkages produces a significant reduction in the
incidence and severity of restenosis. In vitro and animal-model
studies conducted in support of the invention indicate that the
antisense compound (i) is taken up efficiently by cells in a vessel
lumen which are exposed to the antisense compound, (ii) acts
intracellularly to inhibit correct processing (mRNA splicing) and
translation of processed c-myc mRNA, and (iii) is significantly
more effective, in reducing the incidence and severity of
restenosis, than other types of c-myc antisense compounds, e.g.,
phosphorothioate c-myc antisense compounds.
[0080] The synthesis, structures, and binding characteristics of
morpholino oligomers are detailed in above-cited U.S. Pat. Nos.
5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063,
and 5,506,337, all of which are incorporated herein by reference.
The antisense oligomers (compounds) of the present invention are
composed of morpholino subunits of the form shown in the above
cited patents, where (i) the morpholino groups are linked together
by uncharged phosphorus-containing linkages, one to three atoms
long, joining the morpholino nitrogen of one subunit to the 5'
exocyclic carbon of an adjacent subunit, and (ii) the base attached
to the morpholino group is a purine or pyrimidine base-pairing
moiety effective to bind, by base-specific hydrogen bonding, to a
base in a polynucleotide. The purine or pyrimidine base-pairing
moiety is typically adenine, cytosine, guanine, uracil or thymine.
Preparation of such oligomers is described in detail in U.S. Pat.
No. 5,185,444 (Summerton and Weller, 1993), which is hereby
incorporated by reference in its entirety. As shown in the
reference, several types of nonionic linkages may be used to
construct a morpholino backbone.
[0081] Exemplary backbone structures for antisense oligonucleotides
of the invention include the .beta.-morpholino subunit types shown
in FIG. 2A-A through 2D-D. It will be appreciated that a
polynucleotide may contain more than one linkage type.
[0082] Subunit A in FIG. 1 contains a 1-atom phosphorous-containing
linkage which forms the five atom repeating-unit backbone shown at
A-A in FIG. 2, where the morpholino rings are linked by a 1-atom
phosphoamide linkage.
[0083] Subunit B in FIG. 1 is designed for 6-atom repeating-unit
backbones, as shown at B-B, in FIG. 2. In structure B, the atom Y
linking the 5' morpholino carbon to the phosphorous group may be
sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety
pendant from the phosphorous may be any of the following: fluorine;
an alkyl or substituted alkyl; an alkoxy or substituted alkoxy; a
thioalkoxy or substituted thioalkoxy; or, an unsubstituted,
monosubstituted, or disubstituted nitrogen, including cyclic
structures.
[0084] Subunits C-D in FIG. 1 are designed for 7-atom unit-length
backbones as shown for C-C through D-D in FIG. 2. In Structure C,
the X moiety is as in Structure B and the moiety Y may be a
methylene, sulfur, or preferably oxygen. In Structure D the X and Y
moieties are as in Structure B. In all subunits depicted in FIGS.
1A-D, Z is O or S, and P.sub.i or P.sub.j is adenine, cytosine,
guanine or uracil.
[0085] A preferred "morpholino" oligonucleotide is composed of
morpholino subunit structures of the form shown in FIG. 2B-B, where
(i) the structures are linked together by phosphorodiamidate
containing linkages, one to three atoms long, joining the
morpholino nitrogen of one subunit to the 5' exocyclic carbon of an
adjacent subunit, (ii) P.sub.i and P.sub.j are purine or pyrimidine
base-pairing moieties effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X=NH.sub.2, Y=O, and
Z=O.
[0086] As noted above, the compound has a sequence which spans the
start codon of a c-myc mRNA, meaning the compound contains a
sequence complementary to a region of c-myc RNA containing the AUG
mRNA start site and adjacent 5' and 3' base(s). The region of the
mRNA against which the compound is directed is also referred to
herein as the target sequence. The c-myc mRNA to which the
antisense binds may be preprocessed (prespliced) mRNA, in which
case the antisense compound may act to interfere with correct
splicing, leading to truncated forms of the translated protein, or
may bind to the processed mRNA, leading to inhibition of
translation.
[0087] The compound is designed to hybridize to c-myc mRNA, under
physiological conditions with a Tm substantially greater than
37.degree. C., e.g., at least 50.degree. C. and preferably
60.degree. C.-80.degree. C. Although the compound is not
necessarily 100% complementary to the target sequence, it is
effective to stably and specifically bind to the target sequence
such that expression of the target sequence, is modulated. The
appropriate length of the oligomer to allow stable, effective
binding combined with good specificity is about 8 to 40 nucleotide
base units, and preferably about 12-25 base units. Mismatches, if
present, are less destabilizing toward the end regions of the
hybrid duplex than in the middle. Oligomer bases that allow
degenerate base pairing with target bases are also contemplated,
assuming base-pair specificity with the target is maintained. The
compound preferably contains internal 3-base triplet complementary
to the AUG site, and bases complementary to one or more bases 5'
and 3' to the start site. One preferred compound sequence is the
20mer identified as SEQ ID NO:1 and having the base sequence:
5'-ACG TTG AGG GGC ATC GTC GC-3', where the CAT triplet in the
sequences binds to the AUG start site, the 6 bases 3' to the CAT
sequence extend in the upstream (5') direction on the target, and
the 11 bases 5' to the CAT sequence extend downstream on the
target. This compound has enhanced solubility by virtue of having
no self-annealing regions.
[0088] The solubility of the antisense compound, and the ability of
the compound to resist precipitation on storage in solution, can be
further enhanced by derivatizing the oligomer with a solubilizing
moiety, such as a hydrophilic oligomer, or a charged moiety, such
as a charged amino acid or organic acid. The moiety can be
chemically attached to the antisense compound, e.g., at its 5' end,
by well-known derivatization methods. One preferred moiety is a
defined length oligo ethylene glycol moiety, such as
triethyleneglycol, coupled covalently to the 5' end of the
antisense compound through a carbonate linkage, via a piperazine
linking group forming a carbamate linkage with triethyleneglycol,
where the second piperazine nitrogen is coupled to the 5'-end
phosphorodiamidate linkage of the antisense. Alternatively, or in
addition, the compound may be designed to include one a small
number of charged backbone linkages, such as a phosphodiester
linkage, preferably near one of the ends of the compound. The added
moiety is preferably effective to enhance solubility of the
compound to at least about 30 mgs/ml, preferably at least 50 mgs/ml
in aqueous medium.
[0089] The effectiveness of a particular c-myc antisense sequence
may be determined by known screening methods. For example, the
oligomer is incubated with a cell culture expressing the target
RNA, and the presence or absence of the heteroduplex is determined
by techniques such as those set forth in below, or by monitoring
the presence or absence of the encoded, full-length protein as
determined by standard techniques such as ELISA or Western
blotting, or the presence or absence of active protein.
[0090] In another embodiment, the antisense compound forms part of
a particle composition for use in restenosis treatment. One such
particle is a biodegradable particle, e.g., a polylactate or
polyglycolic particle, containing entrapped antisense compound. The
particles are preferably in the 1-5 micron range, and are useful
for delivery by direct particle delivery to an angioplasty vessel
site, as described below, either by being impressed into the vessel
walls by pressure from a balloon against the wall, or by release
from a particle carrier, such as a stent.
[0091] Alternatively, the particles can be microbubbles containing
the compound in entrapped form. The preparation of suitable
microbubbles as antisense carrier is described, for example, by
Porter et al. cited above. The particles may be delivered directly
to the vessel site, that is, by contacting the vessel walls with a
directly with a suspension of the particles, with compound release
from the particles when the vessel region is exposed to ultrasonic
energy.
[0092] In still another embodiment the particles are liposomes
containing entrapped antisense compound. Because the liposome
particles are applied directly to the vessel site, the liposomes
may be conventional liposomes without surface modifications needed
for achieving long circulation times.
III. Method of Treating Restenosis
[0093] Restenosis refers to the renarrowing of the vascular lumen
following vascular intervention, such as coronary artery balloon
angioplasty with or without stent insertion. It is clinically
defined as greater than 50% loss of initial luminal diameter gain
following the procedure. Restenosis is believed to occur in about
30% to 60% of lesions treated by angioplasty and about 20% of
lesions treated with stents within 3 to 6 months following the
procedure.
[0094] "Restenosis" can also occur after a coronary artery bypass
operation, wherein heart surgery is done to reroute, or "bypass,"
blood around clogged arteries and improve the supply of blood and
oxygen to the heart. In such cases, the stenosis may occur in the
transplanted blood vessel segments, and particularly at the
junction of replaced vessels.
[0095] The present invention is directed to methods for reducing
the risk (incidence) or severity (extent of stenosis), particularly
following balloon angioplasty, or in response to other vessel
trauma, such as following an arterial bypass operation. The method
includes administering to the patient, the above-described
antisense compound or composition, in an amount and via direct
local administration of the compound at the vessel site of injury,
to reduce the risk and/or severity of restenosis. In general, an
amount of compound delivered to the vessel site between about 0.5-2
mg antisense compound is preferred, assuming substantially complete
tissue uptake. Thus, where uptake into the vessel tissue is 10% of
amount delivered, the amount delivered is preferably between 5 and
20 mg, preferably in a total volume of between about 0.2 to 1
ml.
[0096] In accordance with one aspect of the method, the modes of
administration discussed below exploits one of more of the key
features: (i) use of an antisense compound that has a high rate of
cell uptake, (ii) the ability of the antisense compound to
interfere with c-myc mRNA processing and mRNA translation, and
(iii) local delivery of the compound by a mode of administration
effective to achieve high localized concentration of the compound
at the vessel injury site. The first two features have been
discussed above. Modes of administration effective to achieve the
third feature will now be detailed.
[0097] A. Iontophoresis
[0098] In one embodiment, the invention provides delivery of the
antisense compound contacting the treated region with a reservoir
containing an antisense compound and introducing the compound from
the reservoir into the vessel by iontophoresis.
[0099] The antisense compounds described herein are uncharged.
Optimal iontophoresis requires that the agent being administered
have an overall net charge. The antisense compounds may be
modified, as described above, to impart at least group that is
charged at physiological or near-physiological pH. Alternatively, a
pulsed electric field may be effective to facilitate the entry of
uncharged antisense compounds into cells through an electroporesis
effect.
[0100] Devices for use in carrying out iontophoretic drug delivery
at a vessel site, e.g., by a balloon-catheter device have been
described. In general, such devices include a reservoir for
compound solution contained in a outer shell of the catheter's
distal-tip balloon, an outer-balloon membrane allowing passage of
the compound from the reservoir to the vessel wall, and an
electrode communicating with the internal reservoir. A second
counter-electrode is placed on the body, and a pulsed voltage is
applied across the two electrodes to create a field that operates
to draw charged compounds into vessel site. Devices, and electric
pulse voltages and times follow those disclosed in the art, e.g.,
Fernandex-Ortiz, Dev, Robinson, and U.S. Pat. Nos. 5,593,974,
5,628,730, and 5,425,703).
[0101] Alternatively, a pulsed-field device designed for diffusion
or injection of uncharged compound into the site, with cell uptake
facilitated by pulsed-field induced electroporation is also
contemplated.
[0102] Both methods provide the advantages of high-efficiency
delivery of antisense compound into the vessel-wall cells, without
the need for high fluid pressure in introducing the compound into
the vessel tissue. Assuming a desired dose of 1 mg for delivery to
the vessel site, and an efficiency of tissue uptake of between
25-80%, the total amount of compound contained in the reservoir for
delivery is between about 1.25 and 4 mg, preferably at a
concentration of between about 25-50 mgs/ml.
[0103] B. Nipple Balloon Catheter or Infiltrator
[0104] In a second general compound-delivery approach, the compound
is injected into the vessel, that it, below the vessel surface, by
means of an injection balloon catheter, such has been described
(e.g., Roy, Pavlides, and Barath). The catheter, which is known
commercially as an "Infiltrator Angioplasty Balloon Catheter" or
"IABC", is a balloon catheter with 3 lumens: one for inflating the
balloon, one central for a guidewire, and a third for drug
delivery. On the surface of the balloon there are several
longitudinal strips or channels, each having a plurality of
injection needles, e.g., six needles, which upon inflation stand
project above the channel surface and are connected to the
drug-delivery lumen. When the balloon inflates, the needles
penetrate the lesion, allowing drug delivery into the tunica media
of the vessel wall.
[0105] In the present invention, the reservoir is filled with an
antisense composition preferably containing a compound
concentration of about 25-50 mgs/ml. Assuming an uptake into tissue
of between about 15-50 percent, the amount of material injected is
in the range of about 0.04 ml to 0.25 ml. The relatively small
volume of compound that is administered reduced the risk of further
injury by fluid injection under pressure into the injury site.
[0106] This mode of administration provides the advantage of high
efficiency of uptake of the compound into the vessel tissue (20% or
greater).
[0107] C. Hydrogel Coating
[0108] In a third delivery approach, the compound is embedded or
dissolved in a diffusable medium, typically hydrogel, that coats
the outer surface of a balloon, e.g., on a balloon catheter used
for angioplasty. Methods for making and using such hydrogel coating
on a catheter balloon have been described (e.g., Imanishi,
Dick).
[0109] The hydrogel coating is formulated to include the antisense
compound, at a preferred concentration of about 25-50 mgs/ml, and
to release the selected dose of the compound for a period of about
5-60 minutes. The total amount of hydrogel is preferably between
about 0.1 to 0.5 ml, allowing a total delivery of about 2.5 to 25
mgs, to accommodate an efficiency of tissue uptake of about
5-40%.
[0110] The hydrogel diffusion method may be combined with
iontophoresis or electroporation, as described above, to enhance
uptake of the compound from the gel into the tissue. In this case,
the amount of material in the gel may be reduced substantially, in
view of the enhanced efficiency of uptake.
[0111] The method has the advantages of maintaining intimate
contact between the compound reservoir and vessel wall during the
compound delivery period, allowing a relatively slow rate of drug
release and uptake by cells, and avoiding elevated injection
pressures.
[0112] D. Stents
[0113] This approach is similar to the hydrogel method above,
except that the compound is contained in diffusable form in a
coating contained on an intravascular stent. The stent may be
placed at the vessel site at the time of balloon angioplasty, or
placed at the site during coronary bypass surgery. Stent designs
and materials, including biodegradable stents which release
compound upon biodegradation, or which include a coating containing
the compound in diffusable form, are known (Raman and U.S. Pat.
Nos. 5,997,468 and 5,871,535).
[0114] As above, the stent or stent coating contains an amount of
drug sufficient to deliver an approximately 0.5-2 mg dose over a
5-60 minute period, with an expected efficiency of uptake into
tissue between 5-20 percent.
[0115] An implanted stent provides two advantages in practicing the
present invention. First, it allows short term dosing, as with the
other methods, and also continued dosing at a lower level over an
extended period, e.g., 1-14 days, to block the early events of
restenosis. Secondly, the stent itself may be effective in reducing
the risk of restenosis, as has been reported.
[0116] E. Microparticles.
[0117] Microparticles, such as polystyrene microparticles (Seradyn,
Indianapolis, Ind.), biodegradable particles, liposomes or
microbubbles containing the antisense compound in releasable form
may be used for direct delivery of the compound into the vessel
tissue.
[0118] The particles are prepared to contain a total dose of
preferably 0.5-2 mg, with the total does depending on the
efficiency of tissue uptake. Where the particles are injected into
the tissue, this uptake will be high, e.g., 30-70% or higher. Where
the particles are merely brought into contact with the vessel wall,
the uptake of compound will be lower.
[0119] Methods for delivery the particles include injection of a
particle suspension, or physical pressing the particles against the
vessel wall, e.g., by balloon pressure in a balloon containing a
outer coating of particles, e.g., in a hydrogel medium, or by
embedding the particles in releasable form in a stent. Where the
particles are microbubbles, the method additional includes exposing
the administered particles to ultrasonic energy to explode the
bubbles and release the bubbles at the particle sites.
[0120] Particle delivery of the compound has the advantage of high
uptake, particular where the particles are injected, and the
potential for both high, short-term drug release and extended
release from depot-release particles, e.g., biodegradable
particles. The particles may also be coated with a binding agent,
e.g., antibodies specific against growth factors or other proteins
that are actively synthesized by endothelial cells during early
cellular events leading to restenosis (see Bauriedel), to enhance
the efficiency of compound uptake. Finally, the antisense compound
may be selectively released from the particles at a desired time,
as in the case for microbubbles.
IV. Restenosis Method
[0121] In a related aspect, the invention includes of treating the
risk of restenosis in a region of a patient's coronary vessel. The
method is carried out by administering to the patient, by local
delivery directly into the region of injury, a morpholino antisense
compound having (i) the base sequence identified as SEQ ID NO:1,
(ii) a phosphorodiamidate backbone shown in. FIG. 2B-B, where
X=NH.sub.2, Y=O, and Z=O, and (iii) a moiety that enhances the
solubility of the compound, preferably to a solubility in aqueous
medium of between 25-50 mgs/ml or greater. The administration is by
direct contact with the vessel, using methods described above, or
alternative methods, such as direct injection of the material
through a Wilinsky type balloon catheter having a drug-solution
reservoir, and means for injecting the solution through pores in
the balloon against the vessel walls. The amount of material
injected is preferably designed to provide a dose of material taken
up by the tissue of between 0.5 to 2 mg antisense compound.
[0122] The moiety that increases compound solubility may be any
biocompatible hydrophilic or charged moiety that can be coupled to
the antisense compound, and which does not interfere with compound
binding to the target sequence. One preferred moiety is a
triethyleneglycol moiety derivatized to the antisense compound
through a carbamate-piperizine linkage as described above.
V. Method of Assaying Effectiveness of Antisense Delivery and
Uptake.
[0123] A standard indicator of the success of PTCA is one or more
follow-up angiograms to determine the minimal lumen diameter of the
affected vessel, that is, the extent of reocclusion. In determining
the success of the methods of the present invention, follow-up
angiograms may be completed one or more times following
implantation of the c-myc antisense-containing catheter. Indicators
of successful therapeutic intervention include a low percent
occurrence of re-occlusion and/or restenosis and a prolonged time
to occurrence of re-occlusion and/or restenosis.
[0124] In accordance with another aspect of the present invention,
there is provided a rapid, easily performed method for confirming
the presence of c-myc antisense compound in target cells, following
antisense administration at the vessel site, and for comparing
uptake levels of the compound achieved by various methods of
compound administration to optimize conditions and dosages for
effective restenosis treatment.
[0125] The method is based on the discovery, disclosed in
above-cited U.S. provisional application 60/117,846 for
"Non-Invasive Method for Detecting RNA", that a morpholino
antisense compound of the type disclosed herein, when administered
in vivo, can be detected in the urine of the receiving subject in a
heteroduplex form consisting of the antisense compound and its RNA
complement. The data indicate a sequence of events that include (i)
uptake of the antisense compound by cells in the subject, (ii)
binding of the compound intracellularly with the target mRNA, (iii)
intracellular nuclease cleavage of single-stranded portions of the
antisense/target complex, leaving a heteroduplex consisting of the
antisense and its mRNA complement; (iv) secretion of the
heteroduplex, presumably recognized as foreign molecules, by the
cells, and (v) appearance of the heteroduplex in the blood and
eventually the urine.
[0126] In the present case, this sequence of events allows for one
to administer c-myc antisense, in accordance with any of the
methods detailed above, and follow the uptake of the compound into
target cells, by monitoring the presence and or quantity of c-myc
antisense/mRNA in the urine or other body fluid, e.g., blood or
serum.
[0127] In practicing the method, the antisense compound of the
invention is administered to a patient or in an animal model in a
selected dose, and by a selected mode of delivery, including any of
the ones mentioned above. Thereafter, and at selected times after
administration, e.g., 4, 12, and 24 hours post administration, the
urine is monitored for the appearance and/or amount of heteroduplex
to determine the effectiveness of compound uptake at the selected
dose and method of administration.
[0128] In one exemplary assay format for use in urine detection, a
sample containing an antisense/:RNA heteroduplex is reacted with a
compound that specifically binds to or modifies the oligomer:RNA
heteroduplex (e.g., a monoclonal antibody (mAb) specific for the
particular heteroduplex) followed by detection of the modified or
conjugated oligomer:RNA heteroduplex.
[0129] In another exemplary assay format, an antisense oligomer is
modified by conjugating it with a reporter molecule before
administration to the subject, followed by separation of
heteroduplexes from uncomplexed reporter labeled antisense oligomer
and detection of the heteroduplex-associated reporter molecule. In
some cases such separation may be carried out by via chromatography
or electrophoresis.
[0130] Exemplary detection methods include spectrophotometric
detection (e.g., with a fluorescence detector), or detection using
antibodies (e.g., FACS analysis). Such methods may be combined with
separation methods in order to expedite analysis, e.g.
chromatographic separation with simultaneous fluorescence detection
or electrophoretic separation with detection by staining of gels,
fluorescence or autoradiographic detection. Such techniques are
known to those of skill in the art and readily adaptable to a given
antisense oligomer and target RNA sequence.
[0131] Any fluorescent molecule known in the art for labeling
nucleic acids may be used in the methods of the invention, for
example, fluorescein and fluorescein derivatives such as carboxy
fluorescein, 5-(4,6-dichlorotriazin-2-yl) amino fluorescein
(5-DTAF); eosin; rhodamines such as Texas Red and
tetramethylrhodamine; cyanine dyes such as thiazole orange, oxazole
yellow and related dyes described in U.S. Pat. Nos. 4,957,870 and
4,888,867; pyrene; porphyrin dyes such as La JollaBlue. The
fluorescent label should be selected such that its fluorescent
lifetime is comparable in magnitude to the correlation time being
measured, taking into account that temperature, viscosity, and the
size of the oligonucleotide to which the fluorescent dye is
conjugated all affect tumbling time. The fluorescent label is
covalently linked or conjugated to the signal primer so as not to
interfere with either emission of fluorescence from the label or
hybridization of the probe to the target sequence. [See, also, U.S.
Pat. Nos. 5,614,617 and 5,652,099.]
[0132] In other cases, antisense oligomers can be synthesized
having a sequence complementary to a given target with the 5' end
of the sequence attached to a reactive amino group as described by
Smith, L. M., et al. Nuc. Acids Res. 13(7):2399 (1985). In such
cases, biotin, peptide or an enzyme, e.g., alkaline phosphatase may
be attached to the 5' amino group. [See, also U.S. Pat. No.
5,783,391.)
[0133] In still another embodiment, the heteroduplex can be
detected, e.g., after isolation from the body-fluid sample, by mass
spectroscopy. In studies conducted in support of the present
invention, it was found that a heteroduplex of RNA:morpholino
oligomer is readily resolved into two different-MW fractions (the
two heteroduplex strands) by mass spectroscopy. This method thus
provides a positive identification of the heteroduplex in terms of
its two component strands.
[0134] As can be appreciated from above, the method allows one to
readily assess the effectiveness of various modes, of
administration, and optimal doses, typically doses that lead to
maximal or near-maximal levels of heteroduplex in the urine. This
will allow a physician to monitor the effectiveness of the
treatment method and assure the physician that the antisense
compound has been taken up by the vessel tissue. If, for example,
the test shows low levels of heteroduplex after 24 hours, the
physician might deem it necessary to retreat the site.
[0135] The following example illustrates the basic features of the
assay method.
EXAMPLE 1
In Vivo Studies with Antisense Oligomer:RNA Heteroduplexes
[0136] Calibration studies performed using an instrument capable of
detecting fluorescein conjugated oligomers (Applied Biosystems
Model 672 GeneScanner) were used to determine the migration rates
of fluorescein-conjugated oligomers of various lengths; a 15-mer, a
20-mer, a 24-mer and a 38-mer ribozyme. Concentrations were
evaluated in a GeneScanner.
[0137] Rats were injected with carboxyfluorescein-conjugated
phosphorodiamidate morpholino oligomers (PMO) which is antisense to
rat cytochrome P-4503A2.
[0138] Chromatograms of plasma samples prepared from blood
withdrawn at the various times post-PMO administration showed the
following. Plasma samples prepared from rats one hour
post-injection contained fluorescent components which migrated at
270 and 340 minutes (two peaks due to the two possible
carboxyfluorescein linkages which migrate differently). Plasma
samples prepared from rats 24 hours post-injection contained
fluorescent components which migrated at approximately 75 and 80
minutes. Mass spectral data (not shown) confirms that the shorter
migration time is not due to degradation of the PMO and indicates
that a PMO:RNA heteroduplex has been formed over that time.
[0139] FIG. 3 represents samples taken at various times (in
minutes) post administration of the P450 antisense PMO, and
indicates the disappearance of the PMO monomer (open squares) and
the corresponding appearance of RNA:PMO heterodimer (solid circles)
in the plasma of rats following such administration. Appearance of
significant quantities of the duplex in plasma does not occur until
the majority of the unduplexed PMO leaves the plasma in what is
generally referred to as the "distribution phase". The PMO
heteroduplex does not accumulate in plasma until after PMO monomer
has distributed into the tissues of the subject where the
complementary mRNA transcripts are localized. The charged RNA:PMO
duplex presumably forms in these tissues and effluxes out of cells
and back into plasma. This overall process requires several
hours.
[0140] After administration of the p450 antisense PMO, fluorescein
was detected in both the kidney and liver. Chromatograms of kidney
tissue sample shows a band at 350 minutes consistent with
unduplexed PMO and an additional band at 80 minutes consistent with
the PMO:RNA heteroduplex, indicating both duplex and parent PMO
which may reside in interstitial spaces or within the cells of the
kidney. The liver tissue sample shows essentially no unduplexed PMO
and significantly more PMO:RNA heteroduplex. These results are
consistent with the observation that levels of P450 mRNA transcript
are much lower in kidney than in liver.
[0141] Studies reflecting the time course of urinary clearance of
unduplexed antisense PMO oligomer and antisense PMO oligomer:RNA
heteroduplexes indicate that several hours are required for
formation and efflux of PMO:RNA heteroduplex from tissues into
plasma, followed by their ultimate appearance in urine.
[0142] Although the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention.
Sequence CWU 1
1
1 1 20 DNA Artificial Sequence antisense 1 acgttgaggg gcatcgtcgc
20
* * * * *