U.S. patent application number 11/093082 was filed with the patent office on 2005-08-04 for near infrared imaging agent.
This patent application is currently assigned to Institut fur Diagnostikforschung GmbH an der Freien Universitat Berlin. Invention is credited to Hilger, Christoph-Stephan, Licha, Kai, Riefke, Bjorn, Semmler, Wolfhard, Speck, Ulrich.
Application Number | 20050169844 11/093082 |
Document ID | / |
Family ID | 6536117 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169844 |
Kind Code |
A1 |
Licha, Kai ; et al. |
August 4, 2005 |
Near infrared imaging agent
Abstract
This invention relates to an in-vivo diagnostic method based on
near infrared radiation (NIR radiation) that uses water-soluble
dyes and their biomolecule adducts, each having specific
photophysical and pharmaco-chemical properties, as a contrast
medium for fluorescence and transillumination diagnostics in the
NIR range, to new dyes and pharmaceuticals containing such
dyes.
Inventors: |
Licha, Kai; (Berlin, DE)
; Riefke, Bjorn; (Berlin, DE) ; Semmler,
Wolfhard; (Glienicke, DE) ; Speck, Ulrich;
(Berlin, DE) ; Hilger, Christoph-Stephan; (Berlin,
DE) |
Correspondence
Address: |
THE WEBB LAW FIRM, P.C.
700 KOPPERS BUILDING
436 SEVENTH AVENUE
PITTSBURGH
PA
15219
US
|
Assignee: |
Institut fur Diagnostikforschung
GmbH an der Freien Universitat Berlin
Berlin
DE
|
Family ID: |
6536117 |
Appl. No.: |
11/093082 |
Filed: |
March 29, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11093082 |
Mar 29, 2005 |
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10368997 |
Feb 19, 2003 |
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10368997 |
Feb 19, 2003 |
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10180272 |
Jun 26, 2002 |
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10180272 |
Jun 26, 2002 |
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09850660 |
May 7, 2001 |
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09850660 |
May 7, 2001 |
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09518947 |
Mar 6, 2000 |
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6258340 |
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09518947 |
Mar 6, 2000 |
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08849369 |
Nov 7, 1997 |
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6083485 |
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08849369 |
Nov 7, 1997 |
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PCT/DE95/01465 |
Jun 6, 1997 |
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Current U.S.
Class: |
424/9.6 ;
548/159; 548/217; 548/305.4; 548/455 |
Current CPC
Class: |
C09B 23/086 20130101;
A61K 49/0017 20130101; A61K 49/0021 20130101; A61K 49/0032
20130101; C09B 69/105 20130101; A61K 49/005 20130101 |
Class at
Publication: |
424/009.6 ;
548/159; 548/217; 548/305.4; 548/455 |
International
Class: |
A61K 049/00; C07D
417/02; C07D 413/02; C07D 043/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 7, 1994 |
DE |
P 44 45 065.6 |
Claims
1-8. (canceled)
9. An in-vivo diagnostic based on near infrared radiation, using
compounds of the general formula I B.sub.L-(F-W.sub.n).sub.m (I)
wherein L represents a number from 0 to 6, n represents a number
from 0 to 10, and m represents a number from 1 to 100; B is a
biological detecting unit having a molecular weight of up to 30,000
that bonds to specific cell populations or selectively to
receptors, or accumulates in tissues or tumours, or generally stays
in the blood, or is a macromolecule that bonds non-selectively; F
represents a dye showing maximum absorption in the range of 650 to
1200 nm; as well as their physiologically tolerable salts,
characterized in that F within the general formula I represents a
cyanine dye of the general formula IIa 10wherein r represents the
numbers 0, 1 or 2, wherein, for r=2, the respective L.sup.6 and
L.sup.7 groups that occur in duplicate may be the same or
different, L.sup.1 to L.sup.7 are the same or different, each
independently representing a CH or CR group, where R is a halogen
atom, a hydroxy, carboxy, acetoxy, amino, nitro, cyano or sulfonic
acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl,
alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or
halogenalkyl group containing up to 6 carbon atoms, an aryl,
alkylaryl, hydroxyaryl, carboxyaryl, sulfoaryl, arylamino,
diarylamino, nitroaryl or halogenaryl group containing up to 9
carbon atoms, or where R represents a bond that bonds to another
group R and forms a 4- to 6-member ring together with the
interspersed groups L.sup.1 to L.sup.7, or two of L.sup.1 to
L.sup.7 that are linked via a --CO-- group, R.sup.3 to R.sup.12 are
the same or different, each independently representing a hydrogen
atom, a group B as defined above or W as defined below, or an alkyl
or alkenyl group containing up to 6 carbon atoms or an aryl or
aralkyl group optionally carrying an additional group W as defined
below, or with 5- to 6-member rings that may be saturated,
unsaturated or aromatic, and that may optionally carry an
additional group R as defined above, annelled to each pair of
adjacent residue R.sup.3 to R.sup.10 with due regard for the
interspersed C atoms, X and Y are the same or different, each
independently representing an O, S, Se or Te or a
--C(CH.sub.3).sub.2--, --CH.dbd.CH-- or --CR.sup.13R.sup.14--
group, where R.sup.13 and R.sup.14 independently represent a
hydrogen atom, a group B as defined above or W as defined below, or
an alkyl or alkenyl group containing up to 6 carbon atoms or an
aryl or aralkyl group containing up to 9 carbon atoms, the alkyl,
alkenyl, aryl or aralkyl group optionally carrying an additional
group W as defined below, characterized in that W within the
general formula I is a carboxy or sulfonic acid group or a
carboxyalkyl group or an alkoxycarbonyl or alkoxyoxoalkyl group
containing up to 12 carbon atoms, and characterized in that W takes
the position of R.sup.4 and R.sup.8 and/or R.sup.6 and R.sup.10
and/or R.sup.11 and R.sup.12.
10. A method of in-vivo diagnosis of disease comprising
administering at least one of the compounds of claim 9 to tissues
and detecting said compound by means of near infrared
radiation.
11. An agent for in-vivo diagnosis comprising at least one of the
cyanine dyes according to claim 9 together with common adjuvants,
substrates and diluants.
Description
[0001] This invention relates to an in-vivo diagnostic method based
on near infrared radiation (NIR radiation) that uses water-soluble
dyes and their biomolecule adducts, each having specific
photophysical and pharmaco-chemical properties, as a contrast
medium for fluorescence and transillumination diagnostics in the
NIR range, to new dyes and pharmaceuticals containing such
dyes.
[0002] Diagnosability of diseases is very much dependent on
obtaining information about the structures, as well as changes, of
tissues of the profound layers that are not primarily accessible.
In addition to palpating, exposing or puncturing these tissues,
such information can be gained using sophisticated imaging methods
such as X-raying, magnetic resonance tomography, or ultrasonic
diagnosis.
[0003] As biological tissue shows a relatively high permeability
for long wave light in the range of 650-1000 nm, a diagnostician
can therefore-use a completely different method of tissue imaging.
The fact that light in the near infrared range can permeate through
several centimetres of tissue is utilized in transillumination
imaging. This technique as yet facilitates diagnosis of
inflammations of the paranasal and maxillary sinuses as well as the
detection of accumulated fluids or blood in superficial zones of
tissue (Beuthan J., Muller G.; Infrarotdiaphanoskopie, Med. Tech. 1
(1992) 13-17).
[0004] Attempts at detecting breast tumours have been
unsatisfactory so far (Navarro, G. A.; Profio, A. E.; Contrast in
diaphanography of the breast; Med. Phys 150 (1988) 181-187;
Aspegren, K.; Light Scanning Versus Mammography for the Detection
of Breast Cancer in Screening and Clinical Practice, Cancer 65
(1990) 1671-77) but there may be better results in the future due
to most recent engineering progress (Klingenbeck J.;
Laser-Mammography with NIR-Light, Gynkol.-Geburtsh.-Randsch 33
Suppl. 1 (1993) 299-300); Benaron D. A.; Optical Imaging reborn
with technical advances, Diagnostic Imaging (1994) 69-76).
[0005] In addition to detecting non-absorbed radiation,
fluorescence radiation emitted after near infrared light treatment
can provide tissue-specific information. This so-called
autofluorescence is used to distinguish atherosclerotic and normal
tissue (Henry, P. D. et al., Laser-Induced Autofluorescence of
Human Arteries, Circ, Res. 63 (1988) 1053-59).
[0006] The main problem of applying near infrared radiation is the
extraordinarily wide scattering of the light which permits-only a
rather blurred image of a clearly contoured object despite
different photophysical properties. The problem increases the
greater the distance from the surface is and may be considered the
major limiting factor of both transillumination and detection of
fluorescence radiation.
[0007] Suitable fluorescent dyes that accumulate in diseased tissue
(above all, in tumours) and that show a specific absorption and
emission behaviour, may contribute towards enhancing the
distinction of healthy from diseased tissue. The change caused by
absorbing irradiated (scattered) light, or fluorescence induced by
exciting radiation, is detected and provides the actual
tissue-specific information.
[0008] Examples of using dyes for in-vivo diagnostics in humans are
photometric methods of tracing in the blood to determine
distribution areas, blood flow, or metabolic and excretory
functions, and to visualize transparent structures of the eye
(ophthalmology). Preferred dyes for such applications are
indocyanine green and fluorescein (Googe, J. M. et al.,
Intraoperative Fluorescein Angiography; Ophthalmology, 100, (1993)
1167-70).
[0009] Indocyanine green (Cardiogreen) is used for measuring the
liver function, cardiac output and stroke volume, as well as the
blood flow through organs and peripheral blood flows (I. Med. 24
(1993) 10-27); in addition they are being tested as contrast media
for tumour detection. Indocyanine green binds up to 100% to albumin
and is mobilized in the liver. Fluorescent quantum efficiency is
low in a hydrous environment. Its LD.sub.50 (0.84 mmol/kg) is great
enough; strong anaphylactic responses may occur. Indocyanine green
is unstable when dissolved and cannot be applied in saline media
because precipitation will occur.
[0010] Photosensitizers designed for use in photodynamic therapy
(PDT) (including haematoporphyrin derivatives, photophrin II,
benzoporphyrins, tetraphenyl porphyrins, chlorines,
phthalocyanines) were used up to now for localizing and visualizing
tumours (Bonnett R.; New photosensitizers for the photodynamic
therapy of tumours, SPIE. Vol. 2078 (1994)). It is a common
disadvantage of the compounds listed that their absorption in the
wavelength range of 650-1200 nm is only moderate. The phototoxicity
required for PDT is disturbing for purely diagnostic purposes.
Other patent specifications dealing with these topics are: U.S.
Pat. No. 4,945,239; WO 84/04665, WO 90/1019, DE-OS 4136769, DE-PS
2910790.
[0011] U.S. Pat. No. 4,945,239 describes a great number of
equipment arrangements for detecting breast cancer using
transillumination and mentions the known fluorescein, fluorescamin,
and riboflavin as contrast-improving absorption dyes. These dyes
share the disadvantage that they absorb in the visible wavelength
range of 400-600 nm in which light transmission capacity of tissue
is very low.
[0012] DE-OS 4136769 describes an apparatus for detecting
fluorescence of tissue areas enriched with fluorescent substances.
These substances are bacterial chlorophyll and its derivatives, and
naphthalocyanines. These structures show absorptions in the range
of 700-800 nm at absorbency indices of up to 70000 l mol.sup.-1
cm.sup.-1. In addition to their fluorescent properties, the
compounds mentioned here are capable of generating singlet oxygen
by radiation, thus having a cytotoxic effect (photosensitizers for
photodynamic therapy). This photosensitizing activity is highly
undesirable for a pure, inactive diagnostic agent.
[0013] Furthermore, synthesis of bacterial chlorophyll compounds is
expensive and requires much effort as natural products have to be
used as parent substances; the naphthalocyanines, however,
frequently show a very low photostability. The known compounds of
these classes are hardly soluble in water, and synthesizing uniform
hydrophilic derivatives is costly.
[0014] WO 84/04665 describes an in-vivo method for the fluorescence
detection of tumours using the following photosensitizers:
haematoporphyrin and its derivative (Hp and HpD), uro- and copro-
and protoporphyrin as well as numerous mesosubstituted porphyrins,
and dyes such as riboflavin, fluorescein, acridine orange,
berberine sulfate and tetracyclines. The photophysical and
pharmacochemical requirements mentioned above are not met by said
substances.
[0015] Folli et al., Cancer Research 54, 2643-2649 (1994), describe
a monoclonal antibody connected with a cyanine dye that was used
for detecting a tumour implanted subcutaneously. Detection of
profounder pathologic processes, however, requires much improved
dyes. Higher dye dosages render the use of antibodies as carriers
unsuitable in view of the side effects to be expected.
[0016] Cyanine dyes and polymethine dyes related to them are also
used as photographic layers. Such dyes need not have any
luminescent properties. Cyanine dyes that have luminescent
(fluorescent) properties have been synthesized for use in
fluorescent microscopy and flow cytometry and coupled with
biomolecules such as compounds containing iodine acetyl groups as
specific labelling reagents for sulfhydryl groups of proteins
(Waggoner, A. S. et al.; Cyanine dye Labeling Reagents for
Sulfhydryl Groups, Cytometry, 10, (1989), 3-10). Proteins are
labelled and isolated in this way. More references: Cytometry 12
(1990) 723-30; Anal. Lett. 25 (1992) 415-28; Bioconjugate Chem. 4
(1993) 105-11.
[0017] DE-OS 39 12 046 by Waggoner, A. S. describes a method for
labelling biomolecules using cyanine and related dyes such as
merocyanine and styryls that contain at least one sulfonate or
sulfonic acid grouping. This specification relates to a single and
two-step labelling method in a hydrous environment, with a covalent
reaction taking place between the dye and the amine, hydroxy,
aldehyde or sulfhydryl group on proteins or other biomolecules.
[0018] DE-OS 3828360 relates to a method for labelling antitumour
antibodies, in particular, antibodies specific to melanoma and
colonic cancer, using fluorescein and indocyanine green for
ophthalmologic purposes. Bonding of indocyanine green to
biomolecules is not covalent (dye-antibody combination,
mixture).
[0019] The known; state-of-the-art methods of in-vivo diagnosis
using NIR radiation thus show a number of disadvantages that
prevented their wide application in medical diagnostics.
[0020] Direct use of visible light or NIR radiation is restricted
to superficial body zones, which is due to the widely scattered
incident light.
[0021] Adding dyes to improve contrast and resolution, however,
gives rise to a number of other problems. The dyes should meet the
requirements that generally apply to diagnostic pharmaceuticals. As
these substances are mostly applied at higher doses and for a
longer diagnostic period, they should be low-toxic. In addition,
dyes suitable for diagnostic purposes should be well soluble in
water and sufficiently stable in chemical and photophysical
respect, at least for as long as the diagnostic period lasts.
Stability as regards metabolization in the system is also
desirable.
[0022] So far, neither dyes nor a suitable method for in-vivo
diagnosis using NIR radiation have been available.
[0023] It is therefore an object of this invention to provide a
method of in-vivo diagnosis that overcomes the disadvantages of
prior art.
[0024] This problem is solved according to the invention by
providing a method of in-vivo diagnosis using NIR radiation in
which compounds of the general formula I
B.sub.l-(F-W.sub.n).sub.m (I)
[0025] are used,
[0026] wherein
[0027] l represents a number from 0 to 6, n a number from 0 to 10,
and m a number from 1 to 100,
[0028] B is a biological detecting unit having a molecular weight
of up to 30000 that bonds to specific cell populations or
selectively to receptors, or accumulates in tissues or tumours, or
generally stays in the blood, or is a macromolecule that bonds
non-selectively;
[0029] F represents a dye showing maximum absorption in the range
of 650 to 1200 nm;
[0030] W represents a hydrophilic group that improves
water-solubility, with the n-octanol-water distribution coefficient
of the compound according to formula I being-less than or equal to
2.0 for l=0;
[0031] as well as their physiologically tolerable salts.
[0032] Compounds of the general formula I that are particularly
well-suited for the method according to the invention are those in
which, for example, B is an amino acid, a peptide, CDR
(complementarity determining region), an antigen, a hapten, an
enzyme substrate, an enzyme cofactor, biotin, a carotinoid, a
hormone, a neurohormone, a neurotransmitter, a growth factor, a
lymphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a
polysaccharide, a dextrane, an oligonucleotide or a
receptor-bonding pharmaceutical.
[0033] Furthermore, such compounds of the general formula I are
particularly appropriate for the method according to the invention
in which, for example, F represents a cyanine dye of the general
formula IIa 1
[0034] wherein
[0035] r represents the numbers 0, 1 or 2, on condition that, for
r=2, the respective fragments L.sup.6 and L.sup.7 that occur in
duplicate may be same or different,
[0036] L.sup.1 to L.sup.7 are same or different, each independently
representing a fragment CH or CR, where
[0037] R is a halogen atom, a hydroxy, carboxy, acetoxy, amino,
nitro, cyano or sulfonic acid, group or an alkyl, alkenyl,
hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl,
alkylamino, dialkylamino or halogenalkyl residue containing up to 6
carbon atoms, an aryl, alkylaryl hydroxyaryl, carboxyaryl,
sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl residue
containing up to 9 carbon atoms,
[0038] or where R represents a bond that bonds to another residue R
and forms a 4- to 6-member ring together with the interspersed
residues L.sup.1 to L.sup.7,
[0039] or where R represents one bond, respectively, at two
different positions that are linked via a --CO-- fragment,
[0040] R.sup.3 to R.sup.12 are same or different, each
independently representing a hydrogen atom, a residue B or W as
defined above, or an alkyl or is alkenyl residue containing up to 6
carbon atoms or an aryl or aralkyl residue containing up to 9
carbon atoms, said alkyl, alkenyl, aryl or aralkyl residue
optionally carrying an additional residue W as defined above,
[0041] or with 5- to 6-member rings that may be saturated,
unsaturated or aromatic, and that may optionally carry an
additional residue R as defined above, annelled to each pair of
adjacent residues R.sup.3 to R.sup.10 with due regard for the
interspersed C atoms,
[0042] X and Y are same or different, each independently
representing an O, S, Se or Te or a --C(CH.sub.3).sub.2--,
--CH.dbd.CH-- or --CR.sup.13R.sup.14-- fragment,
[0043] where R.sup.13 and R.sup.41 independently represent a
hydrogen atom, a residue B or W as defined above, or an alky or
alkenyl residue containing up to 6 carbon atoms or an aryl or
aralkyl residue containing up to 9 carbon atoms,
[0044] the alkyl, alkenyl, aryl or aralkyl residue optionally
carrying an additional residue W as defined above,
[0045] represents a squarain dye of the general formula IIb 2
[0046] wherein
[0047] s and t independently represent the numbers 0 or 1 on the
condition that s and t do not represent the number 1 at the same
time,
[0048] and R.sup.3 to R.sup.12, x and y are as defined above,
[0049] represents a styryl dye of the general formula IIc 3
[0050] wherein
[0051] r, L.sup.1 to L.sup.6, R.sup.3 to R.sup.11 and X are as
defined above,
[0052] or represents a merocyanine dye of the general formula IId
4
[0053] wherein
[0054] r, L.sup.1 to L.sup.6, R.sup.3 to R.sup.8, R.sup.11 and X
are as defined above and G represents an oxygen or sulfur atom.
[0055] Such compounds of the general formula I are particularly
appropriate for the method according to the invention in which, for
example, W is a carboxy or sulfonic acid group or a carboxyalkyl
group or an alkoxycarbonyl group or an alkoxyoxoalkyl group
containing up to 12 carbon atoms,
[0056] represents a residue of the general formulas III
--(CH.sub.2).sub.a--O-Z or (--CH.sub.2--CH.sub.2--O).sub.a-Z
(III)
[0057] wherein
[0058] a represents the numbers 0 to 6
[0059] Z comprises a hydrogen atom or an alkyl residue containing 3
to 6 C atoms that includes 2 to n-1 hydroxy groups, with n being
the number of C atoms or an aryl or aralkyl residue containing 6 to
10 C atoms and carrying 2 to 4 additional hydroxy groups, or an
alkyl residue containing 1 to 6 C atoms and carrying 1 to 3
additional carboxy groups, or an aryl residue containing 6 to 9 C
atoms and carrying 1 to 3 additional carboxyl groups or an aralkyl
residue or a nitroaryl or a nitroaralkyl residue containing 6 to 15
C atoms or a sulfoalkyl residue containing 2 to 4 C atoms carrying
1 to 3 additional carboxy groups,
[0060] or represents a residue of the general formulas IIIa or IIIb
5
[0061] or a residue of the general formula IIIc
--(CH.sub.2).sub.o--(CO).sub.p--NR.sup.1--(CH.sub.2).sub.s--(NH--CO).sub.q-
--R.sup.2 (IIIc)
[0062] wherein
[0063] o and s independently represent the numbers 0, 1, 2, 3, 4, 5
or 6,
[0064] p and q independently represent 0 or 1,
[0065] R.sup.1 and R.sup.2 independently represent a residue Z as
defined above except the substituents of the general formulas IIIa
and IIIb, or independently represent a residue of the general
formulas IIId or IIIe 6
[0066] on the condition that p and q=1,
[0067] or represents a residue of the general formula IIIc as
defined above.
[0068] The compounds used for the method according to the invention
are characterized in that they absorb and fluoresce in the
wavelength range of 650 to 1200 nm, have absorption coefficients of
approx. 100 000 l mol.sup.-1 cm.sup.-1 and more and, where
fluorescence is desirable, have a fluorescence quantum efficiency
greater than 5%, are sufficiently water-soluble, tolerable and
stable in vitro and in vivo as well as photostable. They are
discharged as completely as possible in as short a time as
possible. The compounds used according to the invention are
synthesized easily and at a favourable price in only few reaction
steps from parent materials that are available on the market.
[0069] When applying the method according to the invention in
in-vivo diagnosis, one or several substances of the general formula
I is/are administered to the tissues, for example, by intravenous
injection, then they are irradiated with light from the visible to
the near infrared range of 650 to 1200 nm. Radiation that is not
absorbed and fluorescence radiation are recorded separately or
simultaneously, or against each other with a delay. A synthetic
image is generated from the data obtained.
[0070] Fluorescent images can be recorded using various methods.
Preferred are those methods where the tissue is irradiated
extensively, fluorescence information is visualized in local
resolution by a CCD camera, or where the tissue sectors to be
imaged are scanned by a light ray concentrated in a fibre optical
waveguide and signals is obtained are converted into an image by
computing. The light is beamed in in the narrow-band range at
wavelengths close to the maximum absorption or at
fluorescence-exciting wavelengths of the compounds of the
invention. Radiation that was not absorbed can be recorded as
described, and signals obtained be processed.
[0071] Irradiation angle and angle of observation can be selected
from case to case to meet anatomic and optimum contrast
requirements. The sensitivity of the method may be improved by
subtracting the images prior to and after administering the dye.
Evaluating the time curve of dye-related changes may reveal useful
additional information for the diagnosis.
[0072] The measurement methods used are known to a person skilled
in the art. The expert will also know what equipment parameters
should be set to obtain optimum recording and evaluation conditions
at given absorption or fluorescence wavelengths of the dyes of the
general formula I used according to the invention.
[0073] The compounds of the general formula I used for the method
of the invention cover a wide range of exciting and emission
wavelengths due to the variable structure of the dye system F. It
is possible to gain products with exciting wavelengths that
correspond to a specific source of excitation, e.g. at the diode
laser unit, and are therefore adapted to a given measuring system
or equipment component.
[0074] The techniques described even permit localization of small
objects having a volume of only a few mm.sup.3 at the profounder
layers of tissue or in non-transparent body fluids. Due to light
scattering and the limited resolution it entails it is still
difficult to determined the exact shape and size of such objects
but this is not required to solve some important diagnostic
questions.
[0075] Surprisingly, a fluoroscopic image of a mouse (Swiss nude)
taken after applying a cyanine dye using a CCD camera showed a 1000
times greater fluorescent intensity as compared to a similarly
dosed porphyrin.
[0076] The method described that uses the compounds of the
invention is particularly suitable for the visualization of tissue
without pathological alterations, systemic diseases, tumours, blood
vessels, atherosclerotic plaques, perfusion and diffusion.
[0077] The compounds used according to the invention are applied to
the tissue in different ways. Intravenous administration of the
dyes is particularly preferred.
[0078] Dosage may be quite different depending on the purpose of
application. The goal to be achieved is a detectable concentration
of dye in the tissue zone to be diagnosed, for which a
concentration of 1-100 .mu.g/ml in the tissue or in body fluids
will mostly be sufficient. This concentration is reached by direct
injection into small body cavities or small blood or lymph vessels,
normally by applying 0.1-100 mg of the respective dye contained in
0.1 to 10 ml of vehicle liquid. In this case, 1 to 10 mg of dye are
preferred. Higher doses are mostly required to stain blood vessels
or to detect specific tissues or structures after intravenous
injection (greater than or equal to 100 mg). The upper limit of
dosage is only set by the tolerability of the respective substances
and preparations.
[0079] Thus the invention relates to the use of compounds of the
type B.sub.l-(F-W.sub.n).sub.m, in which F represents a dye from
the class of polymethin dyes, in particular, cyanine dyes.
Merocyanine, styryl, oxonol and squarilium dyes may also be used. W
is a structural element that contributes essentially to the
hydrophilia of the whole molecule. Particularly preferred are
compounds in which l represents the number 0, with their
n-octanol/water distribution coefficient being smaller than 2
(n-octanol/0.01 M TRIS buffer containing 0.9% of sodium chloride,
set to pH 7.4, both phases saturated against each other).
[0080] A biological detecting unit B may, for example, be an amino
acid, a peptide, a CDR (complementarity determining regions), an
antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin;
a carbotinoid, a hormone, neurohormone, neurotransmitter, a growth
factor, a lymphokin, a lectin, a toxin, a carbohydrate, an
oligosaccharide, a polysaccharide, a dextrane, an oligonucleotide
made resistant to nucleases or a receptor-bonding
pharmaceutical.
[0081] Compounds from the above-mentioned groups include, for
example, oxytocins, vasopressins, angiotensins,
melanocyte-stimulating hormones, somatostatins,
tyrotropin-releasing hormones, gonadotropin-releasing hormones,
testosterones, estradiols, progesterones, cortisols, aldosterones,
vitamin D, gastrins, secretins, somatropins, insulins, glucagons,
calcitonine, STH-releasing hormones, prolactins, encephalins,
dopamines, noradrenalines, serotonins, epinephrines, interleucines,
angiogenins, thymopoietins, erythropoietins, fibrinogens,
angiotensinogens, mecamylamines, ranitidin, cimetidin,
lovastatines, isoproterenol derivatives or transferrin.
[0082] These substances facilitate accumulation in specific parts
of the body by targeting the biological detecting unit through
certain mechanisms. These mechanisms include bonding to
extracellular structures, accumulation through various biological
transport systems, recognition of cell surfaces or recognition of
intracellular components.
[0083] Other compounds can be used according to the invention in
which B is a non-selectively bonding macromolecule such as
polylysine, polyethylene glycol, methoxypolyethylene glycol,
polyvinyl alcohol, dextrane, carboxydextrane or a cascade
polymer-like structure that is covalently bonded to F.
[0084] The alkyl-, aryl- or aralkyl residue with hydroxy groups
contained in the compounds of the general formula I used according
to the invention, are for example, 2-hydroxyethyl-,
2-hydroxypropyl-, 3-hydroxypropyl-, 4-hydroxybutyl-,
2,3-dihydroxypropyl-, 1,3-dihydroxyprop-2-yl-,
tris-(hydroxymethyl)-methyl-, 1,3,4-trihydroxybut-2-yl-glucosyl-,
4-(1,2-dihydroxyethyl)phenyl- or 2,4-, 2,5-, 3,5- or
3,4-dihydroxyphenyl residues.
[0085] An alkyl-, aryl- or aralkyl residue containing 1 to 3
carboxy groups may be, for example, a carboxymethyl-,
carboxyethyl-, carboxypropyl-, carboxybutyl-, 1,2-dicarboxyethyl-,
1,3-dicarboxypropyl-, 3,5-dicarboxyphenyl-, 3,4-dicarboxyphenyl-,
2,4-dicarboxyphenyl or 4-(1,2-dicarboxyethyl)-phenyl residue.
[0086] A sulfoalkyl residue preferably is a 2-sulfoethyl-,
3-sulfopropyl- and 4-sulfobutyl residue.
[0087] Compounds in which W takes the position of R.sup.4 or
R.sup.8, R.sup.6 or R.sup.10 and R.sup.11 or R.sup.12, and is also
present in duplicate at positions R.sup.3/R.sup.5 or
R.sup.7/R.sup.9 are particularly preferred.
[0088] The dyes used according to the invention absorb in the
spectral range from 650 nm to 1200 nm. The absorption coefficients
of the compounds are ca. 10000 l mol.sup.-1 cm.sup.-1 and more for
one dye molecule. Fluorescent quantum efficiencies are greater than
5% for all dyes used for fluorescent imaging.
[0089] Another object of this invention are cyanine; dyes of the
general formula V. 7
[0090] where
[0091] Q represents a fragment 8
[0092] where R.sup.30 represents a hydrogen atom, a hydroxy group,
a carboxy group; an alkoxy residue containing 1 to 4 carbon atoms
or a chlorine atom, b is an integer (2 or 3), R.sup.31 represents a
hydrogen atom or an alkyl residue containing 1 to 4 carbon
atoms,
[0093] X and Y independently represent an --O--, --S--,
--CH.dbd.CH-- or --C(CH.sub.2R.sup.32)(CH.sub.2R.sup.33)-- fragment
each,
[0094] R.sup.20 to R.sup.29, R.sup.32 and R.sup.33 independently
represent a hydrogen atom, a hydroxy group, a carboxy-, a sulfonic
acid residue or a carboxyalkyl-, alkoxycarbonyl or alkoxyoxcalkyl
residue containing up to 10 C atoms or a sulfoalkyl residue
containing up to 4 C atoms,
[0095] or a non-selectively bonding macromolecule or a residue of
the general formula VI
--(O).sub.v--(CH.sub.2).sub.o--CO--NR.sup.34--(CH.sub.2).sub.s--(N--CO).su-
b.q--R.sup.35 (VI)
[0096] on the condition that, where X and Y are O, S, --CH.dbd.CH--
or --C(CH.sub.3).sub.2--, at least one of the residues R.sup.20 to
R.sup.29 corresponds to a non-selectively bonding macromolecule or
a compound of the general formula VI,
[0097] where
[0098] o and s equal 0 or independently represent an integer
between 1 and 6,
[0099] q and v independently represent 0 or 1,
[0100] R.sup.34 represents a hydrogen atom or a methyl residue,
[0101] R.sup.35 represents an alkyl residue containing 3 to 6 C
atoms and comprising 2 to n-1 hydroxy groups, with n being the
number of C atoms, or an alkyl residue containing 1 to 6 C atoms
that carries 1 to 3 additional carboxy groups, an aryl residue
containing 6 to 9 C atoms or arylalkyl residue is containing 7 to
15 C atoms, or a residue of the general formula IIId or IIIe 9
[0102] on the condition that q is 1,
[0103] or a non-selectively bonding macromolecule,
[0104] R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and R.sup.26, R.sup.26
and R.sup.27, together with the interspersed carbon atoms, form a
5- or 6-member aromatic or saturated annelled ring,
[0105] as well as their physiologically tolerable salts.
[0106] In the compounds according to the invention of the general
formula V, the alkyl-, aryl- or aralkyl residues containing
hydroxy- or carboxy groups have the preferred composition as
defined above.
[0107] The following are particularly preferred cyanine dyes:
[0108]
5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-(1,2-dicarb-
oxyethyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-i-
ndol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indoli-
um, inner salt, potassium hydrogen salt,
[0109]
2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxym-
ethyl)-1-andecyl)amino]-2-oxcethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-in-
dol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indoliu-
m, inner salt,
[0110]
2-[7-(1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-amino-
]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3--
dimethyl-5-(2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulfobutyl)-
-3H-indolium, sodium salt,
[0111]
2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]--
1,3,5-heptatrienyl]-3,3-dimethyl-5-(Methoxypolyoxyethylene)aminocarbonyl-1-
-(4-sulfobutyl)-3H-indolium, sodium salt,
[0112]
3-(3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-meth-
yl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3-methyl-1-(4-s-
ulfobutyl)-3H-indolium, sodium salt,
[0113]
2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)2H-
-indol-2-yliden]methyl]2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-d-
imethyl-3-ethyl-1H-benz(e)indolium, inner salt,
[0114]
2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-
-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2-
,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-ind-
olium, sodium salt.
[0115] Another important characteristic of the compounds of the
invention and the compounds used according to the is invention is
their hydrophilia marked by a n-octanol/water distribution
coefficient smaller than 2.0 (distribution coefficient
n-octanol/0.01 M TRIS buffer containing 0.9% sodium chloride, set
to pH 7.4, both phases saturated against each other). The compounds
do not have any distinct photosensitizing or phototoxic properties
that would be undesirable in a diagnostic reagent. They are
tolerated well, and discharged.
[0116] The hydrophilic behaviour of the compounds of the invention
make them differ from dyes that have been proposed for use in
in-vivo diagnostics. Especially with cyanine dyes, fluorescent
quantum efficiency values, due to aggregation, drop dramatically in
a hydrous environment, and are comparable to values measured in
non-polar solvents; increased solubility in water and the space
requirements of the hydrophilic groups suppress the formation of
aggregates and micelles.
[0117] A group of preferred compounds shows little protein
affinity; its pharmacokinetic behaviour is similar to that of
insulin or saccharose, for example.
[0118] Surprisingly, these compounds showed diagnostically
sufficient accumulation in specific structures of the system, e.g.
in tumours, despite their simple molecular structure. When the dye
has spread equally throughout the organism, its elimination,
compared to the surrounding tissue, is delayed in tumour zones.
[0119] Tolerance of the substances is very good. Substances having
LD.sub.50 values greater than 0.5 mmol/kg body weight referred to a
single dye molecule, are particularly preferred.
[0120] The compounds of the invention and the compounds used is
according to the invention are characterized by great in-vitro and
in-vivo stability, as well as photostability. When the aqueous
solution is allowed to stand in a daylit room, 98% of each of the
compounds that are particularly preferred show no changes after 2
days, 70% show no changes after 12 days.
[0121] The photophysical and pharmacokinetic properties described
of the compounds of the invention and the compounds used according
to the invention, also differ from those of the only cyanine dye
approved for application in humans: indocyanine green
(cardiogreen).
[0122] Another object of the present invention are compounds of the
general formula I in which the l-values of B are greater than or
equal to 1, preferably 1 or 2.
[0123] Cyanine dyes can be synthesized that have a great extinction
coefficient when absorbing light at wavelengths from 650 to 1200
nm, and that fluoresce with great efficiency. Cyanine dyes of the
invention and cyanine dyes used according to the invention are
mainly synthesized according to methods known from the literature,
for example, F. M. Hamer in The Cyanine Dyes and Related Compounds,
John Wiley and Sons, New York, 1964; Cytometry, 10 (1989) 3-10; 11
(1990) 418-430; 12 (1990) 723-30; Bioconjugate Chem. 4 (1993)
105-11, Anal. Biochem. 217 (1994) 197-204; Tetrahedron 45 (1989)
4845-66, European Patent Appl. 0 591 820 A1.
[0124] The dye-biomolecule adducts of the general formula I used
according to the invention are prepared by reacting a known
compound F-Wn prepared according to the methods mentioned above
with a biological detecting unit B.
[0125] The compound F-W.sub.n should therefore contain at least
one, preferably exactly one, grouping that can react covalently to
an amine, hydroxy, aldehyde or sulfhydryl group on the biological
detecting units. Such groupings are known from the literature and
described in some detail, for example, in DE-OS 39 12 046.
[0126] Particularly preferred are isothiocyanate, isocyanate, and
hydroxysuccinimide ester or hydroxysulfosuccinimide ester groupings
that are reactive to amino functions and form a thiourea, urea and
amide bridge, as well as halogenacetyl and succinimide groupings
that are reactive to sulfhydryl groups and form a thioether
bridge.
[0127] Furthermore, carboxy groups with alcoholic functions may
form ester linkages or ether structures using appropriate
activating reagents (e.g. DCC), and aldehyde functions combined
with hydrazines may result in imine structures.
[0128] The reactive groupings mentioned are added to the dyes of
the invention or dyes used according to the invention of the
general formula I or their synthetic predecessors, or existing
function groups are converted into the reactive groupings. The
reactive groupings may be directly bonded to the dye system via
so-called linker structures (e.g. alkyl chains, aralkyl
structures).
[0129] The F-Wn compounds are preferably reacted with the
biological detecting units B in DMF or a hydrous environment or
DMF/water mixtures at pH values between 7.4 and 10. The molar
proportion of dye and biomolecule (charging ratio) is determined
using absorption spectrometry. Components that are not bound are
separated by chromatography or filtering.
[0130] Macromolecules that have the appropriate function groups may
be coupled to the dyes in a similar way.
[0131] The substances may have quite different properties. Their
molecular weight may be from a few hundreds to more than 10000. The
substances can be neutral or electrically charged. Salts of acid
dyes and physiologically acceptable bases such as sodium, methyl
glutamine, lysine, or salts containing lithium, calcium, magnesium,
gadolinium in the form of cations.
[0132] The dye-biomolecule adducts thus gained excellently meet the
above photophysical, toxicological, chemical and economic
requirements.
[0133] Another object of the present invention is the use of
cyanine dyes of the general formula V for in-vivo diagnosis using
NIR radiation; in analogy to the use of compounds of the general
formula I.
[0134] Yet another object of the present invention are diagnostic
reagents that contain compounds of the general formulas V or I.
[0135] These reagents are produced according to methods known to
persons skilled in the art, optionally by adding common adjuvants,
diluents and the like. This includes physiologically tolerable
electrolytes, buffers, detergents and substances for adjusting
osmolarity and for improving stability and solubility such as
cyclodextrine. Sterility of the preparations during their
production and, in particular, before their application is to be
ensured by taking the steps common in pharmaceutics.
[0136] The invention will now be explained by the following
examples.
EXAMPLES
Example 1
Preparation of
5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-(1,-
2-dicarboxyethyl)amino]-2-oxcethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobut-
yl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3-
H-indolium, inner salt, potassium hydrogen salt
[0137] Di-N-hydroxysuccinimide ester is prepared from
5-carboxymethyl-2-[7-[5-carboxymethyl-1,3-dihydro-3,3-dimethyl-1-(4-sulfo-
butyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl-
)-3H-indolium, inner salt, potassium hydrogen salt according to
known methods (Cytometry 11 (1990) 418-430). 0.16 g (1.22 mmol) of
aspartic acid in 1 ml of DMF are added to a solution of 0.5 g (0.51
mmol) of the disuccinimidyl ester in 5 ml DMF. The reaction mixture
is stirred at room temperature for 48 h. The product is
precipitated by adding ether, purified on RP-18 (LiChroprep,
15-2.mu., H.sub.2O:MeOH 99:1 to 1:1) and lyophilized. 0.27 g (51%)
of product are gained after drying for 24 hours at 50.degree.
C./0.01 mbar.
[0138] Analysis:
[0139] Calc.: C 54.43H 5.54 N 5.40 O 24.68 S 6.18 K 3.77
[0140] Det.: C 54.04H 5.81 N 5.22 S 6.13 K 3.85
Example 2
Preparation of
2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(-
carboxymethyl)-1-andecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-eth-
yl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-
-indolium, inner salt.
[0141] 43 mg (0.65 mmol) of 85% hydrazine hydrate in 1 ml of
methanol are slowly added by dropping to a solution of 0.5 g (0.73
mmol)
2-[7-[5-(carboxymethyl)-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-ylide-
n]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium-N-succini-
midyl ester, inner salt in 5 ml of methanol (Cytometry 11 (1990)
418-430) at -10.degree. C. and stirred for 2 hours at this
temperature. The reaction mixture is evaporated under a vacuum to
approx. 3 ml mixed with 1 ml of isopropanol and kept overnight at
-20.degree. C. The crystals that precipitate are sucked off and
dried using the oil pump. The yield is 0.27 g (61%) of
tricarbocyanine carbonic acid hydrazide.
[0142] 0.27 g (0.45 mmol) of the hydrazide are added under stirring
to a solution of 0.21 g (0.51 mmol) of diethylene triamine
pentaacetic monoethyl ester monoanhydride in 20 ml DMF and 0.2 ml
triethylamine. The mixture is kept agitated at room temperature for
48 hours. The solvent is evaporated at 0.2 mbar after filtering,
the residue is mixed up with CH.sub.2Cl.sub.2, filtered off, and
dried under high vacuum. The product gained is stirred up in 5 ml
of 3M aqueous NaOH at room temperature for 4 hours. Then, a pH
Value of 2.0 is set using semiconcentrated HCl. 1 ml of isopropanol
is added. After allowing the mixture to stand at 4.degree. C. for
18 hours, crystals that have precipitated are sucked off and dried
under high vacuum at 60.degree. C. for 24 hours.
[0143] Yield: 0.23 g (52%) of granulate that glimmers dark red.
[0144] Analysis:
[0145] Calc.: C 59.32H 6.60 N 0.9.88 O 20.96 S 3.23
[0146] Det.: C 54.15H 6.70 N 9.50 S 3.19
Example 3
Preparation of
2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylen-
e)-amino]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrien-
yl]-3,3-dimethyl-5-[2-[[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-su-
lfobutyl)-3H-indolium, sodium salt
[0147] A solution of 0.08 mmol of the N,N-disuccinimidyl ester from
Example 1 in 1 ml of DMF is added to a solution of 800 mg of
methoxypolyoxyethylene amine (ca. 0.16 mmol; average molar weight
ca. 5000) in 10 ml of CH.sub.2Cl.sub.2 and kept agitated at room
temperature for 24 hours. The solid product that precipitates after
adding ether is filtered off and purified by chromatography
(Sephadex G50 medium, H.sub.2O as eluent), yield approx. 58% of
green-blue powder after lyophilization and drying above
P.sub.2O.sub.5.
[0148] average molar weight calc.: 10771, det.: 10820
Example 4
2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-h-
eptatrienyl]-3,3-dimethyl-5-(methoxy-polyoxyethylene)aminocarbonyl-1-(4-su-
lfobutyl)-3H-indolium, sodium salt
[0149] 0.41 g. (0.5 mmol) of
2-[7[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl-
-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-hepatrienyl]-3,3-dimethyl-5-car-
boxy-1-(4-sulfobutyl)-3H-indolium-N-succinimidyl ester, sodium salt
are stirred in an argon atmosphere together with 2.3 g of methoxy
polyoxyethylene amine (0.46 mmol; average molar weight: 5000) in 70
ml CH.sub.2Cl.sub.2 at room temperature for 18 hours. The solvent
is reduced by half under vacuum and the product is isolated as
described in Example 3. The yield is 2.1 g of product in the form
of a green blue powder.
[0150] average molar weight calc.: 5701, det.: 5795
Example 5
Preparation of
3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-0,1,3-dihydro-
-3-methyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3-methyl-
-1-(4-sulfobutyl)-3H-indolium, sodium salt
[0151] 6.5 g (50 mmol) of phenylhydrazine hydrochloride and 8.7 g
(55 mmol) of 5-methyl-6-oxoheptanoic acid are stirred in 50 ml of
concentrated acetic acid at room temperature for 1 hour, and at
120.degree. C. for 5 hours. After reducing by evaporation, the
residue is mixed up with 20 ml of water, and the crystals that have
precipitated are filtered off and dried using the oil pump.
[0152] This yields 9.6 g (83%) of brownish crystals that are
suspended in 60 ml of dichlorobenzene and, after adding 11.6 g (85
mmol) of 1,4-butane sultone, heated for a hours to 150.degree. C.
After the mixture has cooled down to room temperature, 200 ml of
acetone are added, and the precipitate is filtered off. It is
suspended in ether, filtered off again after 18 hours of stirring,
and dried using the oil pump. The yield is 10.7 g (70%) of
3-(3-carboxypropyl)-2,3-dimethyl-1-(4-Sulfobutyl)-3H-indo- lenin
which is purified by chromatography (RP-18, LiChroprep, 15-25.mu.,
MeOH:H.sub.2O as eluent).
[0153] The indotricarbocyanine dye is prepared by heating 5.0 g
(13.6 mmol) of indolenin and 1.9 g (6.8 mmol) of glutaconaldehyde
dianilhydrochloride in 100 ml of acetic anhydride for 30 minutes to
120.degree. C., while adding 25 ml of conc. acetic acid and 2.3 g
(27.6 mmol) of anhydrous sodium acetate. 500 ml of ether are added
to the precipitate gained which is purified by chromatography (in
portions of 1.0 g, RP-18, LiChroprep, 15-25.mu., MeOH:H.sub.2O as
eluent) and finally lyophilized. The yield is 2.5 g (45'-s) of the
final product.
[0154] Analysis:
[0155] Calc.: C 60.13H 6.28 N 3.42 O 19.54 S 7.83 Na 2.81
[0156] Det.: C 59.90H 6.34 N 3.39 S 7.72 Na 2.78
Example 6
Preparation of
2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfo-
butyl).sub.2H-indol-2-yliden]methyl]2-hydroxy-4-oxo-2-cyclobuten-1-yliden]-
methyl]-1,1-dimethyl-3-ethyl-1H-benz(e)indolium, inner salt
[0157] 3.65 g (10.0 mmol) of
3-ethyl-1,1,2-trimethyl-1H-benz(e)indoliumiod- ide are added to a
solution of 1.36 g (8.0 mmol) squaric diethyl ester and 1.6 ml
triethylamine in 12 ml of ethanol that is heated up to 70.degree.
C. After 10 minutes of stirring at 80.degree. C., the mixture is
cooled down to 0.degree. C. The precipitated, red-coloured crystals
are filtered off, washed with ether, and dried under vacuum.
Purification by chromatography on silica gel (CH.sub.2Cl.sub.2:
AcOH 9:1 to 7:3) yields 1.33 g (46%) of
2-ethoxy-1-[(3-ethyl-1,1-dimethyl-1H-benz(e)indol-2-ylide-
n)-methyl]-cyclobuten-3,4-dion.
[0158] This substance is suspended in 15 ml of boiling ethanol and
mixed under stirring with 0.5 ml of 40% NaOH. The solution gained
is stirred for 5 minutes at 80.degree. C. and mixed with 5 ml of 2N
HCl after cooling down to room temperature. The
1-[(3-ethyl-1,1-dimethyl-1H-benz(e)-
indol-2-yliden)-methyl]-2-hydroxycyclobutene-3,4-dion (1.30 g) that
precipitates after evaporating is filtered, dried and used for the
next step of synthesis without being purified.
[0159] The squarain dye is prepared by reacting 1.30 g (3.9 mmol)
of the squaric acid derivative gained with 1.43 g (3.9 mmol) of
3-(3-carboxypropyl)-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolenin. The
components obtained are heated for 18 hours in 80 ml of toluene and
80 ml 1-butanol at the water separator and then freed from solvents
under vacuum. The residue is mixed with ether, and the crystals
that have formed are filtered off after 16 hours of stirring at
room temperature, and purified by chromatography. (RP-18,
LiChroprep, 15-25.mu., MeOH:H.sub.2O as eluent), yield: 0.95 g
(36%)
[0160] Analysis:
[0161] Calc.: C 68.60H 6.20 N-4.10 O 16.40 S 4.70
[0162] Det.: C 68.25. H 6.35 N 4.04 S 4.59
Example 7
Preparation of
2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoet-
hyl]-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]--
5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl-
)-3H-indolium, sodium salt
[0163] 2.0 g (6.4 mmol) of 2,3,3-trimethyl-3H-indol-S-yl acetic
succinimidyl ester in 50 ml CH.sub.2Cl.sub.2 are mixed with 0.84 g
(6.4 mmol) of 4-aminomethyl-2,2-dimethyl-1,3-dioxolane. After 5
hours of stirring at room temperature, the mixture is poured on 100
ml of water and extracted with CH.sub.2Cl.sub.2; the organic phases
are evaporated. After chromatographic purification (silica gel
CH.sub.2Cl.sub.2:MeOH 98:2) 1.86 g (88%) of the amide are gained
which are stirred for 12 hours in 20 ml of dichlorobenzene and 1.36
g (10.0 mmol) of 1,4-butane sultone at room temperature, and at
100.degree. C. The granulate that is formed after stirring the
mixture up with 50 ml of acetone is filtered off and purified by
chromatography (RP-18, LiChroprep, is 15-25.mu., MeOH:H.sub.2O as
eluent). The yield is 0.85 g (28% referred to the parent compound)
of 5-[2-[(2,2-dimethyl-1,3-di-oxa-4-cyclopentyl)methyl]amino-2--
oxoethyl]-2,3,3-trimethyl-1-(4-sulfobutyl-3H-indolenin.
[0164] The reaction to produce the dye is similar to Example 4. The
substance is heated for 10 minutes to 120.degree. C. The crude
product is stirred at room temperature in 5 ml MeOH by adding 100
mg toluene-p-sulfonic acid for 16 hours; insoluble parts are
separated. The filtrate is then kept at -20.degree. C. after adding
3 ml of isopropanol. The powder that precipitates is purified by
chromatography (RP-18, LiChroprep, 15-25.mu., MeOH:H.sub.2O as
eluent), lyophilized and dried for 24 hours at 50.degree. C./0.01
mbar.
[0165] Yield: 0.32 g (37%).
[0166] Analysis:
[0167] Calc.: C 56.70H 6.45 N 5.88 O 20.14 S 6.73 K 4.10
[0168] Det.: C 56.39H 6.88 N 5.67 S 6.58 K 3.93
Example 8
[0169]
2-[7-[1,3-dihydro-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-
-2H-indol-2-yliden)]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxycarbonyl)--
1-(4-sulfobutyl)-3H-indolium, sodium salt at a dose of 3.8
.mu.mol/kg body weight was applied intravenously to an
anaesthesized, tumour-bearing mouse (Swiss Nude, tumour LS 174 T at
the right hindleg).
[0170] The laser-induced fluorescent images were taken prior to,
and at various points in time after, applying the substance with a
fluorescence imager (at Physikalisch-Technische Bundesanstalt,
Berlin Charlottenburg). Radiation was excited using monochromatic
laser light at 740 nm by decoupling the radiation via a fibre
optical waveguide system. Exciting radiation below 740 nm was
removed using a cutoff filter. The laser-induced fluorescence light
above 740 nm was recorded using a CCD camera (Charge Coupled
Device), and data was stored in the form of black-and-white
images.
[0171] The sequence of shots shown in FIG. 1 clearly shows a
general increase of fluorescent intensity after the substance was
applied (A, B). A uniform distribution of intensity can be observed
after 30 seconds, with values being increased in the hepatic and
pulmonary regions and in the tumour (B). With more time elapsing
(up to 1 h) (C,D,E), the substance spreads more and more throughout
the animal. After 18 hours, a clearly increased fluorescent
intensity can be observed in the tumour (right hindleg) as compared
with the rest of the body.
[0172] FIG. 1 shows fluorescent light images (black-and-white) of a
mouse (Swiss Nude) at various points in time after i.v. application
of 3.8 mmol/kg body weight of 2-[7-[1,3
-dihydro-3,3-dimethyl-5-(methoxycarbonyl-
)-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-S-(-
methoxycarbonyl)-1-(4-sulfobutyl)-3H-indolium, sodium salt
[0173] A-E: right lateral images, F: posterior image
[0174] A: prior to application,
[0175] B: 30 secs,
[0176] C: 1 min,
[0177] D: 10 mins,
[0178] E: 1 h after application,
[0179] F: 18 h after application.
* * * * *