Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Diagnostic or test agent produces in vivo fluorescence
Reexamination Certificate
2000-05-01
2003-03-18
Jones, Dameron L. (Department: 1619)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Diagnostic or test agent produces in vivo fluorescence
C424S001110, C424S001650, C424S009100
Reexamination Certificate
active
06534041
ABSTRACT:
DESCRIPTION
The invention relates to acid-labile and enzymatically cleavable compounds for in-vivo and in-vitro diagnosis with near-infrared radiation (NIR radiation), the use of these compounds as optical diagnostic agents and therapeutic agents and diagnostic agents that contain these compounds.
Near-infrared imaging is a non-invasive diagnostic process, in which the high permeability of biological tissue to light on a wavelength 650-1000 nm is exploited. In contrast to the light of the ultraviolet and visible spectral range, which can penetrate only into the topmost millimeter of the tissue, penetration depths into the tissue of up to several centimeters is achieved with use of near-infrared light. The reasons for the basically small penetration depth of light are the absorption of endogenous dyes, mainly hemoglobin and water, which in the spectral range of the near-infrared light, however, have minimum values of between 650 and 1000 nm. This spectral range of the maximum optical tissue transparency is therefore also named a diagnostic/therapeutic window (Boulnois, J., Lasers Med Sci 1986, 1:47-66).
In addition to the modern imaging processes, such as diagnostic radiology, magnetic resonance tomography or ultrasonic diagnosis, another process for graphic tissue visualization is thus available to the diagnostician (Haller, E. B., Time-Resolved Transillumination and Optical Tomography. J. Biomed Optics 1996, 1:7-17).
The use of NIR radiation for site-dependent recording of blood flow and degree of oxygenation in the brains of babies by the detection of the absorption of hemoglobin/deoxyhemoglobin is a process that has been known and used for years (Jöbsis, F. F., Science 1977, 198: 1264-67; Chance, B.; Leigh, J. S.; Miyake, H. et al., Proc Natl Acad Sci USA 1988, 85: 4971-75; Benaron, D. A. et al., Science 1993, 33: 369A.).
The basic problem when near-infrared radiation is used is the strong scattering of light, so that even in the case of different photophysical properties, this object is poorly distinguished from an object with sharp edges and its surrounding area. The problem increases with increasing removal of the object from the surface and can be considered as a main limiting factor both in the case of transillumination and in the detection of fluorescence radiation. As contrast media, dyes, which mark the optical properties of the tissue and result in an increased absorption and fluorescence of the tissue that is to be detected, can therefore make unambiguous detection possible even with poor site resolution. In this case, the absorption behavior of such dye compounds can be used as imaging information. If the dyes, moreover, have the property of emitting the absorbed energy as fluorescence radiation, the latter can also be used as imaging information. In this case, the fluorescence radiation that is red-shifted relative to the excitation radiation is detected separately. The advantage exists, i.a., in that the tissue itself has an extremely low inherent fluorescence in the NIR range and thus the background is minimal. (S. Folli et al., Cancer Research 54, 2643-9 (1994); B. Ballou et al., Cancer Immunol. Immunother. 41, 257-63 (1995); X. Li et al., SPIE Vol. 2389, 789-98 (1995)).
In fluorescence diagnosis, the precondition in this respect is to detect an adequate difference that is as great as possible in the fluorescence emission between the tissue that is to be detected and the surrounding tissue. This can be achieved in principle by a difference in the concentration of the fluorescence dye at a certain time after the substance administration has been achieved. In particular for diagnosis in deeper tissue layers, this difference in the use of substances with unspecific concentration behavior is often inadequate.
The object of the invention is to make available new compounds that overcome the drawbacks of the prior art.
The object is achieved according to the invention by compounds of general formula (I)
(F—L)
m
—A (I),
in which
F stands for a dye molecule with at least one absorption maximum of between 600 and 1200 nm,
L stands for a linker structure, which contains an acid-labile and/or enzymatically cleavable bond,
m is a number between 1 and 80,
whereby if m is a number between 1 and 3,
A represents a dye molecule with at least one absorption maximum of between 600 and 1200 nm, an antibiotically or anticytostatically active molecule, a biomolecule, a non-biological macromolecule or a compound B—(L—W)
o
or D—(L—W)
o
, whereby
D is a non-biological macromolecule,
B is a biomolecule,
L has the above-mentioned meaning,
W represents an antibiotically or anticytostatically active molecule,
o is a number between 1 and 20,
and whereby if m is a number between 4 and 80,
A represents a biomolecule, a non-biological macromolecule or a compound B—(L—W)
o
or D—(L—W)
o
, whereby
D, B, L, W and o have the above-mentioned meanings.
The special property with respect to the in-vivo detection of the near-infrared fluorescence emission of the compounds according to the invention consists in the fact that the latter have little or even no fluorescence emission, and an increase of the fluorescence signal occurs only after this construct is cleaved or after the dye is cleaved off from the construct on the target site (e.g., tumors, inflammations). The effective difference of the fluorescence signal between the tissue that is to be detected and the surrounding tissue is consequently marked by the fact of
a) the concentration difference based on pharmacokinetics mechanisms and
b) by the difference in the fluorescence quantum yield at the time of the diagnosis.
It has been found that the fluorescence of the dyes is quenched when a dye molecule is coupled to another molecule (dimer) while obtaining the compounds according to the invention, i.e., an extremely low fluorescence emission occurs in comparison to the corresponding dye molecule in the unbonded state. It has been found, moreover, that a comparable quenching occurs when other molecules with aromatic structures, which can be both dyes and active ingredients (e.g., cytostatic agents or antibiotic agents), are coupled with the fluorescence dye. Surprisingly enough, a quenching also occurs when the dyes are coupled to the antibodies, antibody fragments and proteins.
In principle, the dyes, which are structural components of the compounds according to the invention, must be distinguished in their monomeric unconjugated form by high molar absorption coefficients and high fluorescence quantum yields.
Preferred compounds of general formula I according to the invention are distinguished in that F and/or A stand for a polymethine dye, tetrapyrrole dye, tetraazapyrrole dye, xanthine dye, phenoxazine dye or phenothiazine dye.
Especially preferred are the structures from the class of polymethine dyes, since the latter have absorption maxima with very high molar absorption coefficients in the near-infrared spectral range of between 700 and 1000 nm (&xgr; up to 300,000 1 mol
−1
cm
−1
), such as, for example, cyanine dyes, squarilium dyes and croconium dyes, as well as merocyanine and oxonol dyes.
Those compounds of general formula (I) according to the invention are also preferred in which F and/or A stand for a cyanine dye of general formula II
in which
R
1
to R
4
and R
7
to R
10
, independently of one another, stand for a fluorine, chlorine, bromine, iodine atom or a nitro group or for a radical —COOE
1
, —CONE
1
E
2
, —NHCOE
1
, —NHCONHE
1
, —NE
1
E
2
, —OE
1
, —OSO
3
E
1
, —SO
3
E
1
, —SO
2
NHE
1
, —E
1
, whereby E
1
and E
2
, independently of one another, stand for a hydrogen atom, a saturated or unsaturated, branched or straight-chain C
1
-C
50
alkyl chain, whereby the chain or parts of this chain optionally can form one or more aromatic or saturated cyclic C
5
-C
6
units or bicyclic C
10
units, and whereby the C
1
-C
50
alkyl chain is interrupted by 0 to 15 oxygen atoms and/or 0 to 3 carbonyl groups and/or is substituted with 0 to 5 hydroxy groups, 0 to 5 ester groups, 0 to 3 carbon groups, 0 to 3 amino g
Licha Kai
Riefke Bjorn
Semmler Wolfhard
Wrasidlo Wolfgang
Institute for Diagnostic Research GmbH of the Free University of
Jones Dameron L.
Millen White Zelano & Branigan P.C.
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