Cyanine and indocyanine dye bioconjugates for biomedical...

Details Cyanine and indocyanine dye bioconjugates for biomedical... Cyanine and indocyanine dye bioconjugates for biomedical...

1999-06-04

2001-04-17

Jones, Dameron L. (Department: 1619)

Drug, bio-affecting and body treating compositions

In vivo diagnosis or in vivo testing

C424S001110, C424S001650, C424S001690, C544S003000, C544S054000

Reexamination Certificate

active

06217848

ABSTRACT:

FIELD OF INVENTION
This invention relates generally to novel dye-bioconjugates for use in diagnosis and therapy. Particularly, this invention relates to novel compositions of cyanine dye bioconjugates of bioactive molecules for site-specific delivery of these agents for optical tomographic, endoscopic, photoacoustic, sonofluorescent, laser assisted guided surgery, and therapeutic purposes. More particularly, this invention relates to a method of preparation and use of cyanine dye bioconjugates for visualization and detection of tumors. This invention is also related to the method of preventing fluorescence quenching by the use of biocompatible organic solvents.
BACKGROUND OF THE INVENTION
Several dyes that absorb and emit light in the visible and near-infrared region of the electromagnetic spectrum are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, or high fluorescence quantum yields. This high sensitivity parallels that of nuclear medicine and permits visualization of organs and tissues without the negative effect of ionizing radiation. Most dyes lack specificity for particular organs or tissues and, hence, these dyes must be attached to bioactive carriers such as proteins, peptides, carbohydrates, and the like to deliver the dyes to specific regions in the body. Several studies on the use of near infrared dyes and dye-biomolecule conjugates have been published (Patonay et al.,1991; Slavik, 1994 Brinkley, 1993; Lee and Woo, U.S. Pat. No. 5,453,505; Hohenschuh, WO 98/48846; Turner et al., WO 98/22146; Licha et al., WO 96/17628; and Snow et al., WO 98/48838). Of particular interest is the targeting of tumor cells with antibodies or other large protein carriers as delivery vehicles (Becker, et al., 1999). Such an approach has been widely used in nuclear medicine applications, and the major advantage is the retention of a carrier's tissue specificity since the molecular volume of the dye is substantially smaller than the carrier. However, this approach does have some serious limitations in that the diffusion of high molecular weight bioconjugates to tumor cells is highly unfavorable, and is further complicated by the net positive pressure in solid tumors (Jain, 1994). Furthermore, many dyes in general, and cyanine dyes, in particular, tend to form aggregates in aqueous media that lead to fluorescence quenching. Therefore, there is a need to prepare low molecular weight dye-biomolecule conjugates to enhance tumor detection, and to prepare novel dye compositions to preserve fluorescence efficiency of dye molecules.
The publications and other materials used herein to support the background of the invention or provide additional details respecting the practice, are incorporated herein by reference, and for convenience are respectively grouped in the appended List of References.
SUMMARY OF THE INVENTION
The present invention relates particularly to the novel composition comprising cyanine dye bioconjugates of general formula 1 wherein a and b vary from 0 to 5; W
1
and X
1
may be
the same or different and are selected from the group consisting of —CR
10
R
11
, —O—, —NR
12
, —S—, or —Se; Q
1
is a single bond or is selected from the group consisting of —O—, —S—, —Se—, and —NR
13
; R
1
, R
10
to R
15
, and R
29
-R
40
may be the same or different and are selected from the group consisting of hydrogen, C
1
-C
10
alkyl, C
1
-C
10
aryl, C
1
-C
10
alkoxyl, C
1
-C
10
polyalkoxyalkyl, —CH
2
(CH
2
OCH
2
)
c
—CH
2
—OH, C
1
-C
20
polyhydroxyalkyl, C
1
-C
10
polyhydroxyaryl, C
1
-C
10
aminoalkyl, —(CH
2
)
d
—CO
2
H, —(CH
2
)
e
—CONH-Bm, —CH
2
—(CH
2
OCH
2
)
f
—CH
2
—CONH-Bm, —(CH
2
)
g
—NHCO-Bm, —CH
2
—(CH
2
OCH
2
)
h
—CH
2
—NHCO-Bm, —(CH
2
)
yy
—OH or —CH
2
—(CH
2
OCH
2
)
zz
—CH
2
—OH; Y
1
is selected from the group consisting of —(CH
2
)
i
—CONH-Bm, —CH
2
—(CH
2
OCH
2
)
j
—CH
2
—CONH-Bm, —(CH
2
)
k
—NHCO-Bm, —CH
2
—(CH
2
OCH
2
)
l
—CH
2
—NHCO-Bm, —(CH
2
)
m
—N(R
14
)—(CH
2
)
n
—CONH-Bm, (CH
2
)
aa
—N(R
29
)—(CH
2
)
bb
—NHCO-Bm, —(CH
2
)
p
—N(R
15
)—CH
2
—(CH
2
OCH
2
)
q
—CH
2
—CONH-Bm, —(CH
2
)
cc
—N(R
30
)—CH
2
—(CH
2
OCH
2
)
dd
—CH
2
—NHCO-Bm, —CH
2
—(CH
2
OCH
2
)
ee
—CH
2
—N(R
31
)—(CH
2
)
ff
—CONH-Bm, —CH
2
—(CH
2
OCH
2
)
gg
—CH
2
—N(R
32
)—(CH
2
)
hh
—NHCO-Bm, —CH
2
—(CH
2
OCH
2
)
ii
—CH
2
—N(R
33
)—CH
2
—(CH
2
OCH
2
)
jj
—CH
2
—CONH-Bm or —CH
2
—(CH
2
OCH
2
)
kk
—CH
2
—N(R
34
)—CH
2
—(CH
2
OCH
2
)
ll
—CH
2
—NHCO-Bm; d, e, g, i, k, m, n, p, aa, bb, cc, ff, hh and yy vary from 1 to 10; c, f, h, j, l, q, dd, ee, gg, ii, jj, kk, ll and zz vary from 1 to 100; Bm is any bioactive peptide, protein, cell, oligosaccharide, glycopeptide, peptidomimetic, drug, drug mimic, hormone, metal chelating agent, radioactive or nonradioactive metal complex, or echogenic agent; Z
1
is selected from the group consisting of —(CH
2
)
r
—CO
2
H, —(CH
2
)
r
—OH, —(CH
2
)
r
—NH
2
, —CH
2
—(CH
2
OCH
2
)
s
—CH
2
—CO
2
H, —CH
2
—(CH
2
OCH
2
)
s
—CH
2
—OH, —CH
2
—(CH
2
OCH
2
)
s
—CH
2
—NH
2
, —(CH
2
)
t
—CONH-Dm, —CH
2
—(CH
2
OCH
2
)
u
—CH
2
—CONH-Dm, —(CH
2
)
v
—NHCO-Dm, —CH
2
—(CH
2
OCH
2
)
o
—CH
2
—NHCO-Dm, —(CH
2
)
w
—N(R
14
)—(CH
2
)
x
—CONH-Dm, (CH
2
)
mm
—N(R
35
)—(CH
2
)
nn
—NHCO-Dm, —(CH
2
)
y
—N(R
15
)—CH
2
—(CH
2
OCH
2
)
z
—CH
2
—CONH-Dm, —(CH
2
)
uu
—N(R
39
)—CH
2
—(CH
2
OCH
2
)
vv
—CH
2
—NHCO-Dm, —CH
2
—(CH
2
OCH
2
)
ww
—CH
2
—N(R
40
)—(CH
2
)
xx
—CONH-Dm,—CH
2
—(CH
2
OCH
2
)
oo
—CH
2
—N(R
36
)—(CH
2
)
pp
—NHCO-Dm,—CH
2
—(CH
2
OCH
2
)
qq
—CH
2
—N(R
37
)—CH
2
—(CH
2
OCH
2
)
rr
—CH
2
—CONH-Dm or —CH
2
—(CH
2
OCH
2
)
ss
—CH
2
—N(R
38
)—CH
2
—(CH
2
OCH
2
)
tt
—CH
2
—NHCO-Dm; r, t, v, w, x, y, mm, nn, pp, uu and xx vary from 1 to 10, and o, s, u, z, oo, qq, rr, ss, tt, vv and ww vary from 1 to 100; and Dm is any bioactive peptide, antibody, antibody fragment, oligosaccharide, drug, drug mimic, glycomimetic, glycopeptide, peptidomimetic, hormone, and the like; R
2
to R
9
may be the same or different and are selected from the group consisting of hydrogen, C
1
-C
10
alkyl, C
1
-C
10
aryl, hydroxyl, C
1
-C
10
polyhydroxyalkyl, C
1
-C
10
alkoxyl, amino, C
1
-C
10
aminoalkyl, cyano, nitro, or halogen.
The present invention also relates to the novel composition comprising cyanine dye bioconjugates of general formula 2 wherein a′ and b′ are defined in the same manner as a and b; W
2
and X
2
are defined in the same manner W
1
and X
1
; Q
2
is defined in the same manner as Q
1
; R
16
is defined in the same manner as R
1
; Y
2
is defined in the same manner as Y
1
; Z
2
is defined in the same manner as Z
1
; and R
17
to R
28
are defined in the same manner as R
2
.
This invention is also related to the method of preventing fluorescence quenching. It is known that cyanine dyes generally form aggregates in aqueous media leading to fluorescence quenching. This problem is further accentuated by the conjugation of large hydrophobic dyes to small molecular peptides. We observed that the addition of a biocompatible organic solvent such as 1-50% dimethylsulfoxide (DMSO) restored the fluorescence by preventing aggregation and allowed the visualization of tumors.
In one embodiment of the invention, the dye-peptide conjugates are useful for optical tomographic, endoscopic, photoacoustic and sonofluorescent applications for the detection and treatment of tumors and other abnormalities.
In another aspect of the invention, the dye-peptide conjugates of the invention are useful for localized therapy.
In yet another aspect of the invention, the dye peptide conjugates of the invention are useful for the detection of the presence of tumors and other abnormalities by monitoring the blood clearance profile of the conjugates.
In a further embodiment of the invention, the dye-peptide conjugates are useful for laser assisted guided surgery for the detection of small micrometastases of, e.g., somatostatin subtype 2 (SST-2) positive, tumors upon laparoscopy.
In yet another aspect of the invention, the dye-peptide conjugates of this invention are useful for diagnosis of atherosclerotic plaques and blood clots.


REFERENCES:
patent: 5453505

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