Note: Descriptions are shown in the official language in which they were submitted.
~ WO 95/19~67 2 ~ 8 ~ ~ 3 ~ I ~"~
THERAPEUTIC TREATMENT FOR INHIBITING
VASCULAR RESTENOSIS
FIELD OF THE INVENTION
This invention relates generally to novel
5 compounds for therapeutic use, and re particularly, to
specific molecularly interactive c..~ u-lds, to methods of
preparin~ and using such specific compounds, and to
pharmaceutical compositions comprising these specific
compounds for therapeutic use in areas of vascular injury,
10 sites of inflammation, vascular atheromatous disease and/or
restenosis .
BACKGROUND OF THE INV ~:IY 11UN
salloon a~gioplasty, atherectomy, rotary ablation
and similar therapeutic techniques used to improve
15 circulation in vivo are finding ever-increasing application
in therapeutic cardiology. Generally, balloon angioplasty
procedures involve the introduction of a balloon-type
catheter into the narrowed portion of an artery. The
narrowing of the artery may be caused by different factors
20 but most commonly is caused by a build-up of
~atherosclerotic plaque~. Once the catheter is positioned
in the narrowed portion of the artery, the balloon portion
of the catheter is inflated. The ;nfl~tirn of the balloon
within the narrowed area of the artery serves to increase
25 the diameter of the blood vessel thus improving
Circ~ t; rln .
Often times, followin~r, a balloon angioplasty
therapeutic procedure or similar therapeutic terhniq~le with
attendant vascular injury, patients experience a re-
30 narrowing or restenosis, of the artery within six monthsaf ter having undergone the angioplasty therapeutic
SUBSrlTU~ SHEET (RULE 26~
WO 95119167 r~
213~S
2.
treatment or after incurring the particular vascular
injury. Restenosis is of considerable concern since its
effects may be life threatening.
Therefore, the need for a suitable ' for
5 therapeutic use to prevent restenosis following balloon
angioplasty or similar therapeutic techni~ues which may
cause vascular injury is of significant importance. It is
an object of the present invention to meet this need.
SU~I~RY OF T~IE INVENTION
The present invention discloses novel
oligonucleotide, peptide, snd polypeptide ~ ~u~ g,
methods of preparing these ~ . ' , ph~ ce~t; cal
compositions comprising these u--ds and the use of
these ~ _ ' - in balloon-type catheters f or therapeutic
15 treatment to inhibit vascular restenosis. Restenosis is a
recurrent stenosis, i.e., a narrowing or stricture of a
duct or canAl. Restenosis and the dev~ of
atheromatous lesions ~the reason for the procedure in the
first place) share several common pathological elements
20 such as the ~ t;~n of monocytes and ma iL~JL,hages at
the area of injury or inf~ n and the proliferation of
vascular smooth muscle. Growth factors which induce this
proliferation of vascular smooth muscle and thus cause
restenosis, arise in large part from the monocytes and
25 macrophages which infiltrate the injured area in response
to infli tory stimuli. The monocytes and macrophages
present in the tissue repre8ent stage5 of differentiation
of the same cell lineage. The cells are referred to as
monocytes when in the blood. Upon deposition in tissue,
30 the cells are called macrophages.
Monocyte Chemotactic Protein-l, hereinafter
SUBSl ITUTE SHEET (RULE 26)
~ WO 95119167 ~ ) 3 ~ JL,~ r-
referred to as MCP-1~ is a member of the C-C' family of
chemo attractant cytokines or rh~ k;n~c~. It is a potent
stimulator of monocyte chemotaxis and has an extremely high
degree of specificity for this cell type. Other family
5 members include Hunlan Macrophage Tnfl; tory Protein-l
uMIP-l) Alpha and Beta, Monocyte Chemotactic Protein-2
~NCP-2~, RANrES, R~ITES precursor and I-309. All of these
r-hA--A,k;n~c incorporate a cysteine-cysteine ~C-C) motif, but
MCP-1 and MIP-l Alpha are the ones most highly specific for
10 monocytes and macrophages. MCP-1 and MIP-l Alpha as well
as the rest of the C-C ~-h ~k; nf~ family are produced by
injured vascular smooth muscle cells. ~he C-C ~h~ k;n~
e . g ., MCP-l so produced attract the monocytes and
macrophages which i nfiltrate the area releasing growth
15 factors and resultir~g in proliferation of vascular smooth
muscle and restenosis.
In using a molecularly interactive th~La~e uLiC
com~ound to inhibit vascular restenosis as discussed
herein, the compou1nd must be highly specific. High
20 specificity, which is essential in such therapeutic
compounds, means t~1at the compound, after having been
introduced into the body, is active to a greater degree
against the target rnolecule or tissue, i.e. the area of
possible restenosis, than on other non-target molecules or
25 tissues. In usirlg oligonucleotides or peptides or
polypeptides as therapeutic compounds, the high specificity
of the particular agent used provides for the strong
ac. 1~t;on or retention of the therapeutic -- ' to
the target molecule or the specific tissue or tissues
30 targeted. In the case of the present invention, the site
of accumulation and retention is in areas of injured
vascular smooth muscle cells as compared with the
ac~ 1~t;~An and retention concentration thereof in other
non-target tissues.
SU13STITUTE SHEET (RULE 2
wogs/1s167 r~"~ s~
218~3
; l
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a balloon-type catheter
such as a balloon infusion catheter i8 coated or filled
with a total, partial or synthetic antisense
5 oligonucleotide or peptide to monocyte chemoattractant
protein ~NCP) material, such as monocyte chemoattractant
protein-l (MCP-l), MIP-l Alpha or other members of the C-C
family of chemotactic cytokines or rh kin~c hereinafter
referred to as antisense NCP-l- or like member of the C-C
10 family of rh~ l~i nf-~ as mentioned above and described in
more detail below. However, for means of simplicity MCP-l
will be used as an example throughout although any other
rl- '-ine family member such as NIP-l Alpha would also be
a suitable target.
An antisense oligonucleotide such as an
antisense oligonucleotide to MCP-l inhibits the trAnc1Atir,n
or transcription of MCP-l mRNA within the vascular smooth
muscle cells or surrounding interstitial space.
Accordingly, MCP-l production is severely inhibited. In
20 the abgence of MCP-l, monocytes ~re not attracted to the
area of vascular injury in their usual numbers. As a
result of the monocytes not infiltrating the area, growth
factors ~GFs) are not released. The relative lack of GFs
does not support the proliferation of vascular smooth
25 muscle cells which cause restenosis in cases of vascular
injury. This is likewise true in the case of antisense
oligonucleotide constructs to NIP-l Alpha, NIP-l Beta,
PANTES, RANTES precursor, and I-309. It may be beneficial
to administer two or more different antisense
30 olig~ rl ~otides or derivatives thereof simultaneously to
inhibit production of two or more cytokines.
SUBST~TUTE SHEET (RULE 26)
~ wo g~/19 167 2 ~ 8 1 ~
. 5
Therapeutic treatment of vascular restenosis can
also be achieved and augmented through the use of another
t of the p~esent invention whereby the antisense
oligonucleotide, to members of the C-C ,h~ -k;ne family,
5 e.g., MCP-l is labelled with a rAtl;nn~lrlide for therapeutic
use. Radiolabelled ;~nt; qPnqe MCP-l r u--ds for
therapeutic use may be cons~ructed using high energy Alpha
or seta emitting i sotopes rather than the pure gamma
emitters customarily used for diagnostic purposes which is
lO also possible and wi ll be discussed in more detail below.
Nature members of the C-C ~h~ nk;n~ family are
produced by post-translational modification of larger
peptides. The sense sequence of the mature MCP-l
polypeptide is as follows:
l 5 NH2 -M G ~ P D A I N A P V T C C Y N F T N R K
S V Q R L A S Y R R I T S S K C P K E A V I F K
T I V A K E I C A D P R Q R W V Q D S N D H L D
R Q T Q T P K T-COOH;
wherein A in each of the examples, repre3ents Alanine, s
20 represents Asparagilrle or Aspartic Acid, C represents
Cysteine, D represents Aspartic Acid, E represents Glutamic
Acid, F ~ ese.lts Pheny1~l~n;n~ G represents Glycine, H
represents Histidine, I represents Isoleucine, K represents
Lysine, L represents Leucine, M represents Methionine, N
25 represents Asparagine, P represents Proline, Q represents
Glutamine, R represents Arginine, S represents Serine, T
represents Threonine, V represents Valine, W represents
Tryptophan, X represents an unspecified or variable amino
acid, Y represents Tyrosine and Z represents Glutamine
3 0 Acid .
SIJBSTITUTE SHEET ~RULE 26)
. .. _ .. . _ .. _ . . . . . . _ _ _
Wo g~/lsl67 P~~ t 1~
21~1~3~ O
.,
The oligonucleotides in the messenger ribonucleic
acid ~mRNA), antisense deo~yribonucleic acid (DNA) and
antisense RNA corresponding to mRNA sequences for MCP-1 are
as follows.
mRNA:
5 ' -AUG CAG CCA GAU GCA AUC AAU GCC CCA GUC ACC UGC
UGU UAU AAC WC ACC AAU AGG AAG AUC UCA GUG CAG AGG
CUC GCG AGC UAU AGA AGA AUC ACC AGC AGC AAG UGU CCC
AAA GAA GCU GUG AUC WC AAG ACC AW GUG GCC AAG GAG
AUG UGU GCU GAC CCC AAG CAG AAG UGG GW CAG GAU UCC
AUG GAC CAC CUG GAC AAG CAA ACC CAA ACU CCG AAG ACU -
3';
Antisense DNA:
5 ' -TAC GTC GGT CTA CGT TAG TTA CGG GGT CAG TGG ACG
ACA ATA TTG AAG TGG TTA TCC TTC TAG AGT CAC GTC TCC
GAG CGC TCG ATA TCT TCT TAG TGG TCG TGG TTC ACA GGG
TTT CTT CGA CAC TAG AAG TTC TGG TAA CAC GGG TTC CTC
TAG ACA CGA CTG GGG TTC GTC TTC ACC CAA GTC GTA AGG
TAC CTG GTG GAC CTG TTC GTT TGG GTT TGA GGC TTC TGA -
3'; and
Antisense RNA:
5 ' -UAC GUC GGU CUA CGU UAG WA CGG GGU CAG UGG ACG
ACA AUA WG AAG UGG WA UCC WC UAG AGU CAC GUC UCC
GAG CGC UCG AUA UCU UCU UAG UGG UCG UCG WC ACA GGG
2 5 WW CW CGA CAC UAG AAG WC UGG UAA CAC CGG WC CUC
UAG ACA CGA CUG GGG WC GUC WC ACC CAA GUC CUA AGG
UAC CUG GUG GAC CUG WC GW UGG GW UGA GGC WC UGA -
3';
wherein ~ lPn;nP, T=Thymine, C=Cytosine, G-GIl~ninp~
U=Ur2cil, B=not A, D=not C, F=not G, K=G or T, M=A or C,
SUBSTITUTE SHEET (RULE 261
W0951191C7 2181~35 F~ll.J,. 5'~[~
.- N=A, C, G or T, R = A or G, S=C or G, V=not T, W=A or T and
Y=C or T.
In targeting antisense oligonucleotides into
smooth muscle cells it is not necP~s~ry that the entire
oligonucleotide se~uence for the mature peptide be present.
Effective complementary binding may reside in a smaller
portion of the molecule. Short segments of antisense
oligonucleotides ma~ be prepared to the mRNA to effectively
block the translation of the mature peptide. The C-C
motif, from which this group of rh~ ~k;noq derives their
name, is a very important structural feature which confers
structural integrity upon the molecule. It is therefore
best to target this area for inhibition of the synthesis of
this class of molecules. For example, peptide sequences
adjacent to the C-C structural motif for members of the ~C-
C- family of ~ kin~q are very effective targets and are
listed below.
The sense MCP-l polypeptide structural motif is
as follows when flanked with five residues on either side
ag referenced in y;~h; - d, T., et al ., FEBS Letters, vol .
244; pp. 487-493 (1~89):
NH~ -N A P V T C C Y N F T R -COOH;
Ant i q~nqe RNA:
5 ' -WA CGG GGU CAG UGG ACG ACA AUA WG AAG UGG WA -
3 '; and
Antisense DNA:
5 ' -TTA CGG GGT CAG TGG ACE ACA A~A TTG AAG TGG TTA -
3'.
SU~STITUI~ SHEE~ (RULE
WO 95/19167 ~ ~ ~ P~ .'C~
2181035
The sense MIP-l Alpha polypeptide structural
motif se~uence is as follows when flanked with five
residues on either side as referenced in Blum, S., et al.,
DNA and Cell Biolo~, Vol. 9; pp. 589-602 (1990):
N~12 -D T P T A C C F S Y T S -COOH;
MIP -1 Alpha Antisense RNA:
5 ' -CUG UGC GGC UGG CGG ACG ACG AAG UCG AUG UGG AGG -
3 '; and
MIP -1 Alpha Antisense DNA:
5 ~ -CTG TGC GGC TGG CGG ACG ACG AAG TCG ATG TGG AGG -
3'.
The sense MIP-l Beta polypeptide structural motif
seguence is as follows when flanked with five residues on
either side:
N~2 -D P P T S C C F S Y T S -COO~I
Antisense RNA:
5 ' -CUR GGN GGN UGN WSN ACR ACR AAR WSN AUR UGN WSN -
3 '; and
Antisense DNA:
5~ -CTR GGN GGN TGN WSN ACR ACR AAR WSN ATR TGN WSN -
3';
wherein R=A or G; N = A, C, G or T/U. W = A or T; S = C or
G.
The sense RANTES ~nd RANTES precursor polypeptide
s~ructural motif sequence is a5 follow9 when flanked with
five residues on either side as referenced in Schall, T.S.,
et al., ~ournal of Immunology, Vol. 141; pp. 1018-1025,
(1988):
SUBSTITUTE SHEET (RULE 26)
~ WO95119167 2181~3~ Pcr/US95~0060~
NH2 -S D q1 T P C C F A Y I A -COOH;
Antisense RNA:
5 ' -AGC CUG UGG UGU GGG ACG ACG AAA CGG AUG UAA CGG -
3'; and
5 Antisense DNA:
5 ' -AGC CTG TGG TGT GGG ACG ACG AAA CGG ATG TAA CGG -
3'.
The sense I-309 polypeptide structural motif
sequence is as follows when flanked with five residues on
lO either side as referenced in Miller, M.D., et al., Journal
of T Inn~Ogy, Vol. 145; pp. 2737--2744 (l990):
NH2 -V P F S R C C F S F A E -COOH;
Antisense RNA:
5 ' -CAU GGG AAG AGG UCU ACA ACG AAG AGU AAA CGC CUC -
3 '; and
Antisense DNA:
5 ' -CAT GGG AAG AGG TCT ACA ACG AAG AGT AAA CGC CTC -
3'.
It may also be usef ul to replace some oxygen atoms in the20 phosphate h~ckhnne with thiol groups to inhibit degradation
in vivo.
In the present invention, the antisense MCP-l
oligonucleotide to a molecule of the C-C r~ ' nf~ family
having similar specificity, may be administered ln vivo
25 using a balloon inf usion catheter with holes in it for
delivery to the particular target site to prevent life-
threatening reste~nosis. The antisense MCP-l
oligonucleotide ma~ also be radiolabeled prior to
administration, usi]~g more than one method. The objective
30 in r~l9;ol~hplin~ is to increase thera~u~ic effect by
SUBSTITUTE SHEET (RU'.E 26~
2 1 8 ~ c ~
bringing this cytostatic properly to bear upon smooth
muscle and to force the cells into apoptosis.
Still another _mhorli t of the present invention
is the introduction of an ~nt;~Pnqe oligonucleotide or the
5 gene for the synthesis of antisense MCP-1 oligonucleotide
into individual vascular smooth muscle cells in area(s) of
vascular injury.
When introducing a gene for the production of an
antisense NCP-1 oligonucleotide into the vascular smooth
10 muscle cells, replication of the antisense MCP-1 iB aided
by placing it under the control of a tissue specific
promoter such as the smooth muscle Alpha actin promoter to
prevent life-threatening vascular restenosis. Vir~l
promoters may also be used such as the cyt~ _ 1 ovirus
15 ~ CNV) promoter .
Such introduction is affected by infusion with a
high concentration of oligonucleotide into the smooth
muscle tissues with a balloon infusion catheter. This
typically requires high ples,5uLe(s) (greater than 2
20 ~ _,h-res) and high c~-~n~ntr~ ,n-- of oligonucleotides
~greater than 12.5 miuLuyL~- per milliliter) and is aided
by 2gents which help to increase the solubility of
-- such as lipid rich liposomes.
If based on antisense or DNA or RNA so as to
25 bind to NCP-l mRNA and prevent translation, the sequence
to be introduced is derived from the antisense or DNA or
P~NA sequences previûusly given on pages 5 through 8.
It is important to nûte that effective inhibition
of translation need not require the entire sequence.
30 Appropriate specificity and 2bility to inhibit may be
SUBSllTUTE SHEET ~RULE 263
~ WO9S/19167 21 81~ r~ . c~
11
- conferred with a seguence of approximately 15 to 30
nucleotides .
As noted above, the cysteine cysteine (C-C) motif
is a common feature characteristic of this family of
S r~h~m~ ;n~ and main~enance of this motif is a critical
factor in preservation of biological activity. Therefore
nucleotide sequences which would inhibit cysteine cysteine
~C-C) translation with preservation of specificity are
particularly effecti-ve. For example the sense mRNA region
l 0 5 ' - AAU GCC CCA GUC ACC UGC UGU UAU AAC WC ACC AAU -3 ',
or the antisense RNA construct 5 ' - WA CGG GGU CAG UGG ACG
ACA AUA WG AAG UGG WA-3 ' which would target the NCP-l
mRNA seguence that stipulates the peptide shown on page 6.
In a furt~1er: ' ~' of this invention, an
15 antisense oligonucleotide was designed to inhibit
translations of both the MCP-l and NIP-l Alpha r~ l~; n~
messages. The designed antisense oligonucleotide seguence
is as follows:
5 ~ -ACA CGA CUG GGG WC CUC WC ACC CAA GUC -3 ' .
This antisense oligonucleotide was designed by
first PY~m;n;n~ the amino acid seguences of MCP-l and NIP-l
Alpha for regions of homology. By using the computer
program MacVector, a high de~ree of homology was observed
between residues 53 through 62 of NCP-l and 55 through 64
in ~P-l Alpha. A stretch of lO residues was chosen so
that the corr~ponl1;nq RNA would consist of 30 bases.
The DNA that codes for both ~CP-l and NIP-l Alpha
has been cloned and reported in the literature. Using the
information, one antisense oligonucleotide that will bind
to the mRNA's coding for both NCP-l and MIP-l Alpha was
Sl~BsllTuTE SHEET (RULE 26~
Wo 95/19167 F~~ Jr~
21810~
12
designed. The above antisense olig~ n~-Cl~otide con~A;nq
only one mismatch with the mRNA for MCP-1 occuring at base
16. C was substituted for G because this purine would not
be able to base-pair with the G at position 16 of mRNA for
5 MIP-1 Alpha because of steric problems. Three mismatches
between the designed antisense oligonucleotide and the mRNA
for MIP-1 Alpha exist. However, some base-pairing should
still occur at these sites because none of the interactions
include two purines, which would cause steric problems.
In a further embodiment of this invention,
therapeutic effects of antisense oligonucleotides upon
potentially proliferating smooth muscle cells are achieved
by radiolAh--lling the antisense MCP-1 oligonucleotide with
a suitable isotope such phosphorous 32 or phrsph~rous 33.
Antisense ~el~tides
An antisense peptide is specified by the DNA
strand ~ 1~ tAry to that which specifies the ordinary
sense peptide. These antisense ~eptides function by
~hydropathic complementarity~ to give binding activity with
its cuL ~ e~uu--ding sense pPr~ er~ and can fllnrtio-~ as
receptor like molecules in af f inity chromatography as
~XP1A;ne~1 by Souza, S.J.U. and Bretani, R. J., Biol. Chem.
267: 13763-13773 (1992). When an antisense peptide is used,
one obtains complementary binding to and inactivation of
the mature MCP-1 polypeptide.
The Ant;~n~e MCP-1 of the present invention is
represented by the following sequence:
NH2 -X G L R X L R G X X T T X L K X L X F X X X
V X X R X X X X X X X X F T G F L R X X K F X X
X R F L X T R L G F V F T X V L X Y L V X L F V
SUBSllTUTE SHEET (RULE 261
Wo 95~19167 PCT/I~S9~10060~
~ 21~ 5
13
- X V X G F X-COOH;
In target:ing mature C-C cytokine family, e.g.,
MCP-l polypeptide with antisense MCP-l polypeptide, it is
not n~c~qs~ry that the complete seventy-six (76) residue
se~auence be present. Effective complementary binding may
reside in a smaller portion of the molecule. Through
substitution in the antisense MCP-l polypeptide se(luence,
and perhaps incorporating ~d) amino acid enantiomorphs,
retroinverse bonds peptidomimetics and the like, additional
useful peptides are developed without affecting
complementary binding specificity and affinity desired.
The reaction in radiol ;~hPl l; ng antisense peptides
generally takes place between the amino groups in the
peptide and the car]~onyl group in the active ester of a
specific ligand to f4rm an amide bond. In particular, the
peptides can be radiolabelled using either a conventional
method referred to as 'post-formed chelate approach' or by
a recent method referred to as ~pre-formed chelate
approach' developed by Fritzberg et al ., U. S . Patent
Numbers 4,965,392 ~nd 5,037,630 incorporated herein by
reference. In the ~pre-formed approach, ' the desired
ligand is complexe~ with the radinn~1cl; ~1~ and then
conjugated to antisense MCP-l polypeptide or a molecule
having antisense MCP-l activity. In the ~post-formed
approach, ~ the desired ligand is first conjugated to the
i3nt;qPnqe peptide and the resulting conjugate is incubated
with the rafl; ~ nl1cl i ~ along with a reducing agent . In the
present invention, t~1e latter approach has the additional
advantage of allowing preparation of the complex in kit
form. Users merely add the radi--nl-cl; c~e to the ligand
antisense MCP-l con iugate or a derivative thereof for
1 ;~hol 1 i n~ to occur .
SUBStlTUTE SHEET (~ULE 26
Wo 95/19167 ~ ~ PCT/US95/00605
2 1 ~
14
It is important to note an unique --^hAn;~m of
the present invention whereby the conjugation reaction will
only occur when the Alpha amino group is in the ~free base~
form, i.e., deprotonated to the NH2 form. If the amino
5 group is protonated, i.e., in the NH3~ form, the reaction
will not occur. Therefore, in the molecules of the present
invention it is potentially important to perform the
conjugation at neutral p~ or within the range of 7.0 to 9.5
to avoid deprotonation of any epsilon-amino groups of
10 lysine, or K. Avoiding the deprotonation of epsilon-amino
groups involved in binding prevents the formation of a
chelate complex which may interfere with the ability of the
antisense peptide to form a complementary complex with MCP-
l. In the present invention, binding preferably occurs on
15 the Alpha amino sJroup in order to avoid potential
interference with the ability of the antisense MCP-l
peptide to form a complementary complex with sense.
Using either method of lAhPl 1 inS~ antisense C-C
~^h L-;nP~, e.g., MCP-l, any suitable ligand can be used to
20 incorporate the preferred r~ ^,nllrl ide metal ion such as
for example but not limited to tP^hnPtium, rhenium, indium,
gallium, samarium, holmium, yttrium, copper, or cobalt, and
more particularly, yttrium-90, rhenium-188, rhPni 186,
indium-lll, te^hnPti~lm-9gm, and derivatives thereof. me
25 choice of the ligand entirely depends on the type of metal
ion desired for th~:Lc-~euLic or even diagnostic purposes.
For example, if the r~ n~cl ;-i~ is a transition element
such as tP~^hnetium or rhenium, then ligands cont~;n;ng
amine, amide, and thiols are preferred to form a stable
30 complex where~s if the rA~ nll~ 1P is a l^nth~nide
element, then polyaminocarboxylates or rhPn~ te type
ligands are preferable.
The above-described uni^yue characteristics of the
SUBSTITUTE SHEET (RULE 26)
~ Wo 95/19167 2 ~ 81 0 3 ~ PCT/USgcl00605
present invention make the radiolabelled antisense MCP-l
polypeptide and its derivatives very attractive for
therapeutic purposes or even diagnostic uses to identify
sites of restenosis and/or vascular injury. The com.pounds
5 of the present irlvention may be labelled with any
radi--n~rl ;.1., favorable for these purposes. Such suitable
radionuclides for radiotherapy include but are not limited
to rhenium-186, copper-67, rhenium-188 and cobalt-60. For
diagnostic purposes the most suitable radionuclides include
10 but are not limited to the transition metals as exem.plified
by te~hn~otillm-99m and copper-62.
Due to the unigue --h;ln; pm em.ployed in the
present invention t:o label the Alpha amino group of
antisense MCP-l peptide and avoid the epsilon amino
15 group(s) (which cou:Ld inhibit the ability of antisense
MCP-l peptides to bind to its complementary sense strand)
a significantly advanltageous radiolabelled peptide compound
for radiotherapy a~ld diagnostic imaging of areas of
potential restenosis is achieved.
As previously noted, a preferred: ' ~';- t of
the present inventio~ is the antisense peptide, polypeptide
or protein to MCP-l or derivatives thereof used alone to
prevent vascular restenosis. However, additional
c of the present invention include antisense MCP-
l or derivatives thereof radiolabelled using a pre-formed
or post-formed --~ho~ o9y.
In a pref erred : ' ' t according to the present
invention, an antisense C-C cytokine, e.g., MCP-l or a
molecule having sense MCP-l interactive capability is first
bonded to the N3S aminothiol ligand which is illustrated in
Figure 1
SUBSllTUlE SHEET (RULE 26)
.
WO 9~/19167 P
21v~3~
16
~1N--\
lC~.
~¢, ~
Figure 1
wherein m is a whole number less than eleYen and preferably
3; p is either 0 or 1; PGI is a suitable sulfur protecting
group selected from the group consisting of C1 20 S-acyl such
5 as alkanoyl, benzoyl and substituted benzoyl -whereby
alkanoyl is preferable, C1_2D S-~cyl groups such as benzyl,
t-butyl, trityl, 4-methoxybenzyl and 2,4-dimethu~yLell~yl -
whereby 2,4-~i h~xybenzyl is preferable, Cl 10 alkoxyalkyl
such as methoxymethyl, ethoxyethyl and tetrahydLu~
10 -whereby tetrahydropyranyl is preferable, carbamoyl, and C~
~0 alkoxyc~rbonyl such as t-butoxycarbollyl and
methoxycarbonyl -whereby t-butoxycarbonyl is preferable;
and X is a coupling moiety selected from the group
consisting of carboxyl, amino, isocyanate, isothiocyanate,
15 imidate, maleimide, chluLuua~ Lv--yl, chlorosulfonyl,
succinimidyloxycarbonyl, h~loacetyl and C~ ~0 N-
alku~yuaLL ,yl -whereby N ~~ h~ cylcabamoyl is preferable.
In another pref erred : ' ~ '; t according to the
present invention, antisense MCP-1 or a molecule having
20 sense MCP-1 interactive rr.r~h;1;ty is bonded to the N2S2
aminothiol ligand which is illustrated in Figure 2;
r ~
Figure 2
SUBSTlTUrE SHEET (RULE 26)
W09~;119167 2~ 81~35 F~~ t.-~'
17
- wherein n is a whole number less than eleven 2nd preferably
3; PG2 and PG3 may be the same or different sulfur
protecting s~roups selected from the group consisting of Cl-20
S-acyl such as alkanc~yl, benzoyl and substituted benzoyl
5 whereby alkanoyl is preferable, Cl 20 alkyl groups such as
benzyl, t-butyl, 4-methoxybenzyl, trityl and 2,4-
~1; thr~xybenzyl -whereby 2,4-~ h~ybenzyl is preferable,
Cl lo alkoxyalkyl such as for example methoxymethyl,
ethoxyethyl, ancl tetrahydropyranyl -whereby
lO tetrahydropyranyl i:s preferable, cA ' ~1 and Cl lO
alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl and
t-butoxycarbonyl -whereby t-butoxycarbonyl is preferable;
and Y is a couplillg moiety selected from the group
consisting of carboxyl, amino, isocyanate, isothiocyanate,
15 imidate, maleimide, chlorocarbonyl, chlorosulfonyl,
SU~r;nim; dyloxycarbonyl, h~1O,A cetyl, and Cl lO N-
alkoxycarbamc~yl -whereby N-methoxylcabamoyl is preferable.
In another pref erred embodiment of the present
invention, an antisense C-C cytokine, e.g., to MCP-l or a
20 molecule having interactive CAr~qh; l; ty with sense ~CP-l is
conjugated with the ligand illustrated in Figure 3,
~ b
f < ~^
Figure 3
wherein n varies from 1 to 10, and Y is a coupling moiety
selected from the group consisting of carboxyl, amino,
25 isocyanate, isothioganate, imidate, maleimide,
chluL.,~ alb~ l, chlorosulfonyl, s~c~-;n;m;rlyloxycarbonyl,
SUE~ TUlE SHEET (RU~E 26~
WO 95/19167 , PCT/US95/00605
2181Q3~ ~
18
haloacetyl, and Cl lO N-alkoxycarbamoyl such as N-
methoxycarbamoyl and t-butoxycarbamonyl -whereby t-
butoxyrA ~ yl is preferable; and R is selected from the
group consisting of hydrogen and C~ ~0 alkyl such as methyl
5 and t-butyl -whereby t-butyl is preferable.
In another preferred embodiment, an antisense C-C
rh It-;n~, e.g., MCP-l or a molecule having interactive
rAr~h; l; ty with sense MCP-l can be conjugated with the
metal complex illustrated in Figure 4
.~ ,o
or/-o~~
Figure 4
wherein m is a whole number less than eleven and more
preferably 3; p is either 0 or 1; X' is a coupling moiety
selected from the group consisting of carboxyl, amino,
isocyanate, isothiocyanate, imidate, maleimide,
15 chlorocarbonyl, chlorosulfonyl, suc;n;n;m;dyloxycarbonyl,
haloacetyl and C~ lO N-alkoxycarbamoyl such as N-
methoxyrA ' ~y 1 and t -butoxycarbamoyl -whereby
t-butoxycarbamoyl is preferable and ~ is a r~A;~n~lrl ;Ae
suitable for diagnostic imaging or therapeutic use such as
20 technetium, rhenium, copper, cobalt, indium, gallium,
samarium, yttrium and holmium.
In another preferred G hoA; t, an antisense C-C
r~ ;ne, e.g., MCP-l or a molecule having interactive
ri9rAhjl;ty with sense MCP-l can be conjugated with a metal
25 complex as illustrated in Fi51ure 5 wherein Y' and n are
defined the same respectively as Y and n in Figure 3 and N
is def ined the same as M in Figure ~ .
SUBSrlTUTE SHEET (RULE
~ wo 95/191~7 2 1 ~ f ~ C
., 19
.- ~
Figure 5
In another ]?referred embodiment, an antisense C-C
k;n~, e.g., MCP-l or a molecule having interactive
c~p~h;1;ty-with sense MCP-l can be conjugated with a metal
5 complex as shown in Figure 6.
~o~
Figure 6
wherein Z', g and R are defined the same respectively as Y,
n and R of Figure 3 and M is defined the same as M in
Figure 4.
In another preferred e~l~o~l;m~nt, an 2ntisense C-C
~h ~k;n~ e.g., MCP-l or a molecule having interactive
hil;ty with sense NCP-l can be conjugated with a metal
complex as shown in Figure 7.
~ ~
SuBSTmJTE SHEET (RULE 26)
WO 95/19~67 . ~ 51t~ "'
2181~5
~ Figure 7
wherein M is def ined the same as M in Figure 4 .
Common esters which have been found useful in
this 1 ~hPl 1; ng techni~ue are o- and p- nitrophenyl, 2 -
5 chloro-4-nitrophenyl, cyanomethyl, 2 ~ opyridyl,
hydroxybenztriazole, N-hydroxysucr;n;m;~ trichlorophenyl,
t et ra f luorophenyl, t hi opheny l, t et ra f luorothiophenyl,
o-nitro-p-sulfophenyl, N-hydroxyphthAl ;m;-le and the like.
For the most part, the esters will be formed from the
10 reaction of the carboxylate with an activated phenol,
particularly, nitro-activated phenols, or a cyclic _lou.ld
based on hydroxylamine.
me advantages of using sulfur protecting groups
include the fact that a separate step for removal of the
15 sulfur-protective group is not ner~ssAry. me protecting
groups are displaced from the compound during the ~h~tl;ng
in what is believed to be a metal-assisted acid cleavage:
i.e., the protective groups are ~ P1AC~P~1 in the presence
of a radionuclide at an acid pH and the rA-l; nn~lrl; dP is
20 bound by the rhPlat;ng ~. me rAtl;olilhpl;nr~
~lc,cedule thus is simplified, which is a significant
advantage when the chelating _ u.~dx are to be
radiol~hPlle-l in a hospital laboratory shortly before use.
Additionally, another advantage of the present invention is
25 that the basic pH conditions and harsh conditions
~soc; ~ted with certain known radiol ~hPl; n~ procedures or
pl.,ced~Les for removal of other sulfur protected groups are
avoided. mus, base-sensitive groups on the chelating
compounds survive the radio-l i~hPl l; n~ step intact .
30 Suitable sulfur-protecting groups, when taken together with
the sulfur atom to be protected, include hemithioacetal
groups such as ethoxyethyl, tetrahydrofuranyl,
SUBSTIME SHEET (RULE 26)
WO9SI19167 P( ~ .C S~!~
2~ 5
21
methoxymethyl, and tetrahydropyranyl. Other suitable
sulfur protecting groups are Cl ~O acyl groups, preferably
alkanoyl or benzoyl. Other possible formulas for the
chelating compounds are described in U. S . Patent Number
4, 965, 392 incorpora~ed herein by reference.
Synthesis of the rr~ nu~l i rl~ bifunctional
chelate and subsequent conjugation to antisense MCP-l, or
a derivative thereof, can be performed as described in U.S.
Patent Number 4,965,392 incorporated herein by reference
10 and related technologies as covered by U.S. patent numbers
4,837,003, 4,732,974 and 4,659,839, each incorporated
herein by ref erence,
After purification, the radiolabelled antisense
C-C ~h~ ;n~, e.g., MCP-l, or derivatives thereof, may be
15 injected into a patient or theL~uLic use or even
diagnostic imaging depending on the ra~l; on~ rle used.
The radiolabelled antisense ~CP-l compound of the present
invention is capabl~ of radiotherapeutic use or reliably
visllAl;7;n~ areas of poten~ial restenosis within minutes
20 post-injection. The antisense MCP-l peptide when
r~i ol AhPl ~ ed with the Re-186 or Re-188 triamide thiolate
bifunctional chelate is particularly efficacious as an in
vivo radiotherapeutic agent for areas of restenosis.
~ach of the ~ of the present invention
25 are described in still greater detail in the illustrative
examples which follow:
ExamDlQ 1:
Antisense RNA or DNA or a derivative thereof for
purposes of inhibition of translation is prepared by
30 oligonucleotide synthesis using the solid phase
phosphotrizster method detailed by Woods, et al., Proc.
Natl. Acad. Sci. USA, Vol. 79; pp. 5661-5665 (1982) and
SUBS~Ill)TE SHEET (r.ULE 26)
Wo 9~/19167 ~ 1 8 1 0 3 ~ 5~
22
suspended to a concentration of between lO and 500
mi~ LC~ . per milliliter in lOmM Tris chloride with lmM
ethyl~nP~l;Am;n-~tetraacetic acid (EDTA) and infused into the
lesion using a balloon infusion catheter at pressures of
two to eight atmospheres. Contact time should be in the
range of 5 to 30 minutes. If it is desired to radiolabel
the preparation with phosphorus -32 or ~hns}~hnrus-33 to
increase therapeutic effect, ~hn~qr~hnrus-32 or phosphorus-33
labeled nucleotides are prepared using the methods giYen by
Maxam, A.~. /and Gilbert, W., Pro. Natl. Acad. Sci. USA,
Vol. 75; pp. 560-564 (1977).
~x~ple 2:
A solution of antisense MCP-l peptide, or
derivatives thereof, ~O.Ol mmol) in 2 mL of
carbonate/bicarbonate buffer at pH 8.5 i 0.5 is treated
with a solution of 0 . l mmol of the ligand illustrated in
Figure l (wherein m=2, p=l, PG1 is benzoyl, and X is
succinimidyloxycarbonyl) in dimethylfn~m;-le (0.5 mL) and
the entire mixture is kept at room temperature for 2 hours.
The mixture is then diluted with water ~2.5 mL) and
dialyzed extensively against water. After dialysis, the
solution is lyophilized to give the desired ~nt;q~nqe ~CP-l
conjugate .
gx~ 3:
A solution of antisense ~ICP-l peptide, or
derivatives thereof, 10 . Ol mmol ) in 2 mL of
carbonate/bicarbonate buffer at pH 8.5 i 0.5 is treated
with a solution of 0 . l mmol of the ligand illustr2ted in
Figure 2 (wherein n=2, PG2 and PG3 are benzoyl, and Y is
sllr~r;n;m;~9yloxycarbonYl) in dimethylfor-~m;~P (0.5 mL) and
the entire mixture is kept at room temperature for 2 hours.
The mixture is then diluted with water (2.5 mL) and
dialyzed extensively against water. After dialysis, the
SUBSTITUIE SHEET (RULE 26)
wo 95/19167 2 ~ 8 ~ Q 3 5 r~
23
solution is lyophili:~ed to give the desired antisense MCP-l
conjugate .
Examvl~ 4:
A solution of antisense MCP-l peptide, or
5 derivatives thereof, (O.Ol mmol) in 2 mL of
carbonate/bicarbonate buf f er at pH 8 . 5 i 0 . 5 is treated
with a solution of O . l mmol of the ligand illustrated in
Figure 3 ~wherein g=4, and Z is succinimidyloxycarbonyl) in
dimethylformamide (0.5 mL) and the entire mixture is kept
lO at room temperature for 2 hours. The mixture is then
diluted with water (2 . 5 mL) and dialyzed extensively
against water. After dialysis, the solution is lyophilized
to give the desired antisense MCP-l conjugate.
ExaD~DlQ 5:
To lOO uL of a solution containing 5 mg of sodium
gluconate and O . l mg of stannous chloride in water, 500 U1
of 99m-TcO4 (pertechnetate) is added. After incubation at
room temperature for about lO minutes, a solution of 500 uL
of the antisense MCP-l polypeptide, or derivatives thereof,
20 conjugates (l mg/mL in O.l M carbonate/bicarbonate buffer,
pH 9 . 5 ) as described in Examples l or 2 is then added and
the entire mixture is incubated at 37C for about l hour.
The desired labelled peptide is separated from unreacted
99mTc-gluconate and o~ her small molecular weight impurities
25 by gel filtration chromatography tSephadex G-50) using
phosphine buffered ph~siological saline, (hereinafter PBS),
0.15M NaCl, pH 7.4 as eluent.
ExamDl~ 6:
A mixture of gentisic acid (25 mg), inositol ~lO
30 mg), and the antisense MCP-l polypeptide, or derivatives
thereof, conjugate (500 uL, l mg/mL in water) was treated
with In-lll indium chloride in O . 05 } HCl. me solution
SllBS~ITUTE SHEET (RULE 2~)
Wo 95/19167 PcT/Usss/0060~
2~ 81~35
24
was allowed to incubate at~ room temperature for about 30
minutes. The desired labèlled peptide is separated from
unreacted In-111 indium salts and other small molecular
weight impurities by gel filtration chromatography
5 (Sephadex G-50) using rhn5ph;ne. buffered physiologic~1''
saline, (PsS), 0.15~ NaCl 25 eluent.
~xampl~ 7:
Antisense DNA or a derivative thereof for
purposes of inhibition of MCP-1 synthesis by inhibition of
10 transcription by self replication within smoo~ muscle
cells is prepared by introduction of such DNA sequences
into a plasmid (a circular piece of DNA) consisting of a
smooth muscle actin or viral promoter coupled to antisense
DNA to MCP-1 and appropriate start ~nd stop sign&ls. This
15 plasmid is introduced into smooth muscle cells by using a
balloon infusion catheter. The plasmid DNA is z~ ed to
cnnt Pntration of between 10 and 100 mi- L-aSI per
milliliter in Tris chloride EDTA ~10 IrM, 1 m~l ETDA~ (TE)
and is infused at a pLt:S~UL~:: of between 2 and 8
20 al ~oreS. Infusion time varies between 5 snd 30
minutes .
After the Ant; R-~nRe MCP-1 polypeptide,
oligonucleotide or a derivative thereof is prepared and
optionally labelled according to the l~LoceduL~ de~Rcribed
25 above, the compound is used with a rhAr~--p~lt;cally
~ 'rtAhl e carrier in a method of performing ther2py or
radiotherapy or a method of performing a diagnostic imaging
procedure using a 8amma camera or like device. These
procedures involve injecting or administering, for example
30 by means of a balloon injector catheter, to a warm-blooded
animal an effective amount of the present invention and
then in the case of diagnostic use, ~Ypos; ng the warm-
blooded animal to an imaging procedure using a suitable
SIJBSIITUTE SHEET (RULE 26~
W0 95/19167 P. ~ r-
218~ ~35
detector, e.g. a gamma camera. Images are obtained by
recording emitted radiation of tissue or the pathcloqical
process in which the radioactive peptide or oligonucleotide
has been incorporated, which in the present case are
5 potential sites of restenosis, thereby imaging at least a
portion of the body of the warm-blooded animal.
phAr~cPlltically acce]?table carriers for either diagnostic
or therapeutic use include those that are suitable for
injection . or admini.stration such as aSIueous buffer
10 solutions, e.g. tris (hydroxymethyl)aminomethane (and its
salts), chloride phosphate, citrate, bicarbonate, etc.,
sterile water for injection, physiological saline, and
balanced ionic sol~tions con~A;n;n~ chloride and/or
bicarbonate salts of normal blood plasma cations such as
15 Ca~, Na', K~ and ~g2~ Other buffer solutions are described
in Reminqton's Pr~ctice of PharmacY, 11th edition, for
example on page 170. The carriers m.~ay contain a rhPl~t;ng
agent, e.g. a small amount of ethyl~n~;Am;netetrAAret;c
acid (EDTA), calcium, disodium salt, or other
20 rhArm~ceutically acceptable rh~ t;ng agents.
The concent~ation of the 1 AhPl 1 ed or llnl Ahel 1 ed
peptide and the pharmaceutically acceptable carrier, for
example in- an aS~ueous medium, varies with the particular
field of use. A sufficient amount is present in the
25 pharmaceutically acceptable carrier in the present
invention when satisfactory visllAl;7At;rn of areas of
vascular injury is achievable or satisfactory therapeutic
results are achievable.
The composition is administered to the warm-
30 blooded animals so t3~at the composition remains in the
living animal for about six to seven hours, although
shorter and longer residence periods are normally
acceptable .
SUBSTITUTE SHEET (RULE 2
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . . . . _ . ... .. _
WO 95/19~67 P~ ~
~181~3~ --
26 ~
~ `
The antisense MCP-1 compounds of the present
invention or antisense MCP-1 derivative thereof, prepared
as described herein, provide means of in vivo therapeutic,
radiotherapeutic or diagnostic imaging of areas of
5 potential restenosi8.
After consideration of the above specification,
it will be appreciated that many imp, c,v~ - c and
; f; CA~ nR in the details may be made without departing
from the spirit and scope of the invention. It i9 to be
10 understood, therefore, that the invention is in no way
limited, except a~ defined by the Ap~-n~ claims.
SUBST~TUrE SHEET (RULE 26)
