CA2181035A1

CA2181035A1 - Therapeutic treatment for inhibiting vascular restenosis

Info

Publication number: CA2181035A1
Application number: CA002181035A
Authority: CA
Inventors: Leon R. Lyle; Beth Thomas-Miller
Original assignee: Individual
Current assignee: Mallinckrodt Inc
Priority date: 1994-01-14
Filing date: 1995-01-13
Publication date: 1995-07-20
Also published as: JPH09508358A; EP0802792A1; WO1995019167A1

Abstract

A composition suitable for administration to a warm-blooded animal comprising an antisense oligonucleotide to the C-C chemokine family typified by MCP-1 and MIP-1-Alpha which may or may not be labeled with a radionuclide by means of a chelate ligand capable of administration to an animal to produce reliable visual imaging of areas of potential restenosis or to produce therapeutic effects on areas of potential restenosis.

Description

~ 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

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~

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~

- 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~

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:

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 - 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 ~

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~

~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.-~' - 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~ ~

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 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~

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~

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 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)

Claims

We claim:

1. The oligonucleotide defined by antisense sequence 5'-ACA CGA CUG GGG UUC CUC UUC ACC CAA GUC-3'.

2. A composition suitable for administration to a warm-blooded animal comprising said oligonucleotide of claim 1 or a derivative thereof to inhibit translation of mRNA for members of a C-C chemokine family typified by MCP-1 and MIP-1 Alpha, so as to inhibit vascular restenosis.

3. A method of in vivo vascular therapy, comprising administering to a warm-blooded animal a therapeutically effective amount of the oligonucleotide of claim 1 or a derivative thereof to inhibit translation of mRNA for members of C-C chemokine family typified by MCP-1 and MIP-1 Alpha so as to inhibit vascular restenosis.

4. The oligonucleotide of claim 1 or a derivative thereof capable of inhibiting vascular restenosis upon in vivo administration.

5. The oligonucleotide of claim 1, 2, 3 or 4 wherein said oligonucleotide is labeled with Phosphorus -32 or Phosphorus -33 and is capable of administration to a warm-blooded animal to inhibit vascular restenosis.

6. A plasmid construct consisting of the oligonucleotide of claim 1 or a derivative thereof linked to a smooth muscle actin or viral promoter capable of replication within smooth muscle cells to produce therapeutic effects on restenosis.

7. A composition suitable for administration to a warm-blooded animal comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof to inhibit vascular restenosis.

8. A method of in vivo vascular therapy, comprising administering to a warm-blooded animal a therapeutically-effective amount of the oligonucleotide of claim 1 an antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof to inhibit vascular restenosis.

9. The oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof capable of inhibiting vascular restenosis upon in vivo administration.

10. The oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof, wherein said oligonucleotide or peptide is labeled with a radionuclide by means of a chelate capable of administration to a warm-blooded animal to inhibit vascular restenosis.

11. A therapeutic composition suitable for administration to a warm-blooded animal comprising said oligonucleotide of claim 1 labeled with Re-186 or Re-188 by means of a triamide thiolate (N3S) chelate capable of administration to an animal to produce therapeutic effects on areas of restenosis.

12. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal a therapeutically-effective amount of said oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof labeled with Re-186 or Re-188 by means of a triamide thiolate (N3S) chelate to allow for therapeutic effects on areas of restenosis.

13. The oligonucleotide of claim 1 labeled with Re-186 or Re-188 by means of a triamide thiolate (N3S) chelate.

14. The oligonucleotide of claim 1 of claim 13 wherein said antisense oligonucleotide labeled with Re-186 or Re-188 Re by means of a triamide thiolate (N3S) chelate is capable of administration to a warm-blooded animal to produce therapeutic effects on areas of restenosis post-administration.

15. A diagnostic composition suitable for administration to a warm-blooded animal comprising said oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor RANTES, I-309 peptide or a combination of two or more thereof or derivative thereof labeled with a suitable radionuclide by means of a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate capable of administrationto an animal to produce reliable diagnostic imaging of areas of potential restenosis.

16. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an imaging-effective amount of said oligonucliotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof labeled with a suitable radionuclide by means of a triamide thiolate (N3S) or diamide dithiolate (N2S2) chelate to allow for diagnostic imaging of areas of potential restenosis.

17. The oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivative thereof labeled with a suitable radionuclide by means of a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate.

18. The oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof, wherein said oligonucleotides or peptides are labeled with a suitable radionuclide by means of a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate capable of administration to a warm-blooded animal to produce reliable diagnostic imaging of areas of potential restenosis within two and one half hours post-injection.

19. A therapeutic composition suitable for administration to a warm-blooded animal comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof labeled with a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate bound to a suitable radioactive isotope capable of administration to an animal to produce therapeutic effects on areas of potential restenosis.

20. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal a therapeutically-effective amount of the oligonucleotide of claim 1 antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivative thereof labeled with a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate bound to a suitable radioactive isotope to produce therapeutic effects on areas of potential restenosis.

21. The oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 peptide or a combination of two or more thereof or derivatives thereof labeled with a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate bound to a radioactive isotope.

22. A composition suitable for administration to a warm-blooded animal comprising an antisense oligonucleotide with MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES or I-309 interactive capability capable of administration to an animal to produce therapeutic effects on areas of potential restenosis.

23. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal therapeutically-effective amount of an antisense oligonucleotide with MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES or I-309 interactive capability to produce therapeutic effects on areas of potential restenosis.

24. An antisense oligonucleotide with MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES or I-309 interactive capability to therapeutically inhibit vascular restenosis upon administration to a warm-blooded animal.

25. The antisense oligonucleotide with MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES or I-309 interactive capability of claims 23, 24, or 25 wherein said peptide labeled with a radionuclide by means of a triamide thiolate (N3S) or a diamide dithiolate (N2S2) chelate is capable of administration to a warm-blooded animal to produce therapeutic effects on areas of potential restenosis.

26. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 or a combination of two or more thereof or derivatives thereof which retains MCP-1 interactive capability conjugated with a N3S ligand having the general structure Figure 1 wherein m is a whole number less than eleven; p is either 0 or 1; PG1 is a sulfur protecting group selected from the group consisting of C1-20 S-acyl, C1-20 alkyl, C1-10 alkoxyalkyl, carbamoyl and C1-10 alkoxycarbonyl and X is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl.

27. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivatives thereof conjugated with a N2S2 ligand having the general structure Figure 2 wherein n is a whole number less than eleven; PG2 and PG3 may be the same or different sulfur protecting groups selected from the group consisting of C1-20 S-acyl, C1-20 alkyl, C1-10 alkoxyalkyl, carbamoyl and C1-10 alkoxycarbonyl and Y is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl.

28. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivatives thereof conjugated with a phenolic ligand having the general structure Figure 3 wherein n is a whole number less than eleven; Y is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl; and R is hydrogen or a C1-10 alkyl.

29. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivatives thereof conjugated with a metal complex having the general structure Figure 4 wherein m is a whole number less than eleven; p is either 0 or 1; X' is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl; and M is technetium, rhenium, indium, yttrium, gallium, samarium, holmium, copper or cobalt.

30. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivative thereof conjugated with a metal complex having the general structure Figure 5 wherein Y' is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl; n is a whole number less than eleven; and M is technetium, rhenium, indium, yttrium, gallium, samarium, holmium, copper or cobalt.

31. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES
precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivatives thereof conjugated with a metal complex having the general structure Figure 6 wherein q is a whole number less than eleven; wherein Z' is a coupling moiety selected from the group consisting of carboxyl, amino, isocyanate, isothiocyanate, imidate, malaeimide, chlorocarbonyl, chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl and C1-10 N-alkoxycarbamoyl; R is selected from the group consisting of hydrogen, and C1-10 alkyl; and M is technetium, rhenium, indium, yttrium, gallium, samarium, holmium, copper or cobalt.

32. A composition comprising the oligonucleotide of claim 1, antisense MIP-1 Alpha, MIP-1 Beta, RANTES precursor, RANTES, I-309 or an antisense molecule having MCP-1 interactive capability or a combination of two or more thereof or derivatives thereof conjugated with a metal complex having the general structure Figure 7 wherein M is technetium, rhenium, indium, yttrium, gallium, samarium, holmium, copper or cobalt.

33. The composition of claim 26 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

34. The composition of claim 27 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

35. The composition of claim 28 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

36. The composition of claim 29 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

37. The composition of claim 30 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

38. The composition of claim 31 labelled in a 99mTc-pertexhnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

39. The composition of claim 32 labelled in a 99mTc-pertechnetate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

40. The composition of claim 26 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

41. The composition of claim 27 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

42. The composition of claim 28 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

43. The composition of claim 29 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

44. The composition of claim 30 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

45. The composition of claim 31 labelled with 111In-indium derivatives such as indium chloride, citrate or tartarate.

46. The composition of claim 32 labelled with 111In-indium derivatives such as indium chloride, or tartarate.

47. The composition of claim 26 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

48. The composition of claim 27 labelled in a 186/188 Re-perrheneate golution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

49. The composition of claim 28 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

50. The composition of claim 29 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and 2. transfer ligand such as sodium gluconate or tartarate.

51. The composition of claim 30 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

52. The composition of claim 31 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

53. The composition of claim 32 labelled in a 186/188 Re-perrheneate solution containing a reducing agent, a buffering agent, and a transfer ligand such as sodium gluconate or tartarate.

54. The composition of claim 26 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

55. The composition of claim 27 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

56. The composition of claim 28 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

57. The composition of claim 29 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

58. The composition of claim 30 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

59. The composition of claim 31 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

60. The composition of claim 32 labelled with 90Yt derivatives such as yttrium chloride, citrate or tartarate.

61. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 33 for diagnostic imaging of areas of potential restenosis.

62. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 34 for diagnostic imaging of areas of potential restenosis.

63. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 35 for diagnostic imaging of areas of potential restenosis.

64. A method of performing a diagnostic pocedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 36 for diagnostic imaging of areas of potential restenosis.

65. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 37 for diagnostic imaging of areas of potential restenosis.

66. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 38 for diagnostic imaging of areas of potential restenosis.

67. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 39 for diagnostic imaging of areas of potential restenosis.

68. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 40 to produce therapeutic effects on areas of potential restenosis.

69. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 41 to produce therapeutic effects on areas of potential restenosis.

70. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 42 to produce therapeutic effects on areas of potential restenosis.

71. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 43 to produce therapeutic effects on areas of potential restenosis.

72. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 44 to produce therapeutic effects on areas of potential restenosis.

73. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 45 to produce therapeutic effects on areas of potential restenosis.

74. A method of performing a therapeutic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 46 to produce therapeutic effects on areas of potential restenosis.

75. The composition of claim 33, wherein M is 99?technetium.

76. The composition of claim 34, wherein M is 99mtechnetium.

77. The composition of claim 35, wherein M is 99mtechnetium.

78. The composition of claim 36, wherein M is 99mtechnetium.

79 The composition of claim 37, wherein M is 99mTechnetium.

80. The composition of claim 38 wherein M is 99mTechnetium.

81. The composition of claim 39 wherein M is 99mTechnetium.

82. The composition of claim 40, wherein M is indium-111.

83. The composition of claim 41, wherein M is indium-111.

84. The composition of claim 42, wherein M is indium-111.

85. The composition of claim 43, wherein M is indium-111.

86. The composition of claim 44, wherein M is rhenium-186 or rhenium-188.

87. The composition of claim 45, wherein M is rhenium-186 or rhenium-188.

88. The composition of claim 46, wherein M is rhenium-186 or rhenium-188.

89. The composition of claim 47, wherein M is rhenium-186 or rhenium-188.

90. The composition of claim 48, wherein M is rhenium-186 or rhenium-188 .

91. The composition of claim 49 wherein M is rhenium-186 or rhenium-188.

92. The composition of claim 50, wherein M is rhenium-186 or rhenium-188.

93. The composition of claim 51, wherein M is rhenium-186 or rhenium-188.

94. The composition of claim 52, wherein M is rhenium-186 or rhenium-188.

95. The composition of claim 53 wherein M is rhenium-186 or rhenium-188.

96. The composition of claim 54, wherein M is yttrium-90.

97. The composition of claim 55 wherein M is yttrium-90.

98. The composition of claim 56, wherein M is yttrium-90.

99. The composition of claim 57, wherein M is yttrium-90.

100. The composition of claim 58, wherein M is yttrium-90.

101. The composition of claim 59, wherein M is yttrium-90.

102. The composition of claim 60, wherein M is yttrium-90.

103. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 61 to image areas of potential restenosis.

104. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 62 to image areas of potential restenosis.

105. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 63 to image areas of potential restenosis.

106. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 64 to image areas of potential restenosis.

107. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 65 to image areas of potential restenosis.

108. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 66 to image areas of potential restenosis.

109. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 67 to image areas of potential restenosis.

110. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 68 to image areas of potential restenosis.

111. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 69 to image areas of potential restenosis.

112. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 70 to image areas of potential restenosis.

113. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 71 to image areas of potential restenosis.

114. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 72 to image areas of potential restenosis.

115. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 73 to image areas of potential restenosis.

116. A method of performing a diagnostic procedure, which comprises administering to a warm-blooded animal an effective amount of the composition of claim 74 to image areas of potential restenosis.

117. A composition suitable for administration to a warm blooded animal comprising an antisense MCP-1 peptide or a derivative thereof to inhibit vascular restenosis.