Diagnostic substances for optical imaging testing on the basis of nanoparticular formulations

Abstract

The present invention relates to the provision of nanoparticular formulations comprising a PEG-alkyl block copolymer and a near infrared fluorescent dye, the preparation of these nanoparticular formulations, pharmaceutical compositions comprising the nanoparticular formulations of the present invention, as well as their use as contrast medium.

Claims

1. A micellic formulation, comprising 1) a compound (1) comprising polyethylene glycol or methoxypolyethylene glycol as hydrophilic structural element and an alkyl chain as lipophilic structural element, wherein the alkyl chain has 3 to 30 carbon atoms, which are optionally independently mono- or poly-substituted with C.sub.1-C.sub.3 alkyl, hydroxyl or phenyl, and wherein the hydrophilic and the lipophilic structural elements are connected via a covalent bond, which covalent bond is an ether, ester, amid, carbamate, thiocarbamate, thioether, or urea bond, and 2) a near infrared fluorescent dye (2), which is indocyanine green or a derivative of indocyanine green, in which derivative of indocyanine green a) one or two sulfobutyl chains at the indole nitrogen are substituted with —C.sub.1-6-alkyl-R.sup.2, wherein R.sup.2 is selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+, —NH.sub.2, —N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, —C≡C, —C.sub.1-20-alkyl, —CONH—C.sub.1-20 alkyl, —NHC(O)—C.sub.1-20 alkyl and —O—C.sub.1-20 alkyl, wherein the C.sub.1-20 alkyl is a branched or straight-chain alkyl in which one or more non-consecutive methylene units can be substituted with a unit selected from the group consisting of O, S, NH, C(O)NH, SO.sub.2, SO, aryl, ethene and ethine, and wherein the alkyl is substituted with at least one group selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+, —NH.sub.2, —N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, and —C≡C; and/or b) the polymethine chain is substituted with a substituted polymethine chain with a group R.sup.3 at the central carbon atom, wherein the two adjacent carbon atoms can form a 5- or 6-membered ring together with the three carbon atoms of the polymethine chain, wherein R.sup.3 is selected from the group consisting of —C.sub.1-6-alkyl-R.sup.2, -phenyl-C.sub.1-6alkyl-R.sup.2, —S-phenyl-C.sub.1-6alkyl-R.sup.2, —O-phenyl-C.sub.1-6alkyl-R.sup.2, and —O-phenyl-C.sub.1-6alkyl-R.sup.2, wherein R.sup.2 is defined as above; and/or c) the outer benzindole rings are substituted with one or more groups independently selected from the group consisting of —SO.sub.3.sup.−Na.sup.+, —COOH and —OH, wherein micelles have been formed in an aqueous media from the combination consisting of only the compound (1) and the near infrared fluorescent dye (2), wherein the diameter of the micelles is in a range of 1 nm to 50 nm, and wherein the fluorescence of the micelles is in a range of 750 to 900 nm.

2. The micellic formulation according to claim 1, wherein the alkyl chain is derived from a saturated, unsaturated or chemically or biochemically modified fatty acid.

3. The micellic formulation according to claim 1, wherein the near infrared fluorescent dye is a derivative of indocyanine green, in which derivative of indocyanine green a) one or two sulfobutyl chains at the indole nitrogen are substituted with —C.sub.1-6-alkyl-R.sup.2, wherein R.sup.2 is selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+, —NH.sub.2, —N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, —C≡C, —C.sub.1-20-alkyl, —CONH—C.sub.1-20 alkyl, —NHC(O)—C.sub.1-20 alkyl and —O—C.sub.1-20 alkyl, wherein the C.sub.1-20 alkyl is a branched or straight-chain alkyl in which one or more non-consecutive methylene units can be substituted with a unit selected from the group consisting of O, S, NH, C(O)NH, SO.sub.2, SO, aryl, ethene and ethine, and wherein the alkyl is substituted with at least one group selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+, —NH.sub.2, —N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, and —C≡C; and/or b) the polymethine chain is substituted with a substituted polymethine chain with a group R.sup.3 at the central carbon atom, wherein the two adjacent carbon atoms can form a 5- or 6-membered ring together with the three carbon atoms of the polymethine chain, wherein R.sup.3 is selected from the group consisting of —C.sub.1-6-alkyl-R.sup.2, -phenyl-C.sub.1-6alkyl-R.sup.2, —S-phenyl-C.sub.1-6alkyl-R.sup.2, —O-phenyl-C.sub.1-6alkyl-R.sup.2, and —O-phenyl-C.sub.1-6alkyl-R.sup.2, wherein R.sup.2 is defined as above; and/or c) the outer benzindole rings are substituted with one or more groups independently selected from the group consisting of —SO.sub.3.sup.−Na.sup.+, —COOH and —OH.

4. The micellic formulation according to claim 1, wherein the near infrared fluorescent dye is indocyanine green.

5. A pharmaceutical composition comprising the micellic formulation according to claim 1 and a pharmaceutically acceptable carrier.

6. A contrast medium suitable for in vivo administration, comprising the micellic formulation according to claim 1, which formulation is in a form suitable for in vivo administration.

7. A process for preparing the micellic formulation according to claim 1, comprising dissolving the compound comprising polyethylene glycol or methoxypolyethylene glycol as hydrophilic structural element and an alkyl chain as lipophilic structural element in water, and adding the fluorescent dye to the solution, wherein a micellic formulation is formed.

8. The micellic formulation according to claim 1, wherein the alkyl chain has been hydroxylated, epoxidated, acetylated, carboxylated or esterified.

9. The micellic formulation according to claim 1, wherein the alkyl chain is a hydroxy stearic acid.

10. The micellic formulation according to claim 1, wherein the polyethylene glycol is a polyethylene glycol fatty acid ester block copolymer.

11. The micellic formulation according to claim 1, wherein the covalent bond is an ether, ester, amid, carbamate, thiocarbamate, or thioether bond.

12. The micellic formulation according to claim 1, wherein the hydrophilic structural element is polyethylene glycol.

13. The micellic formulation according to claim 1, wherein the hydrophilic structural element is methoxypolyethylene glycol.

14. The micellic formulation according to claim 1, which has a hydrodynamic diameter of 12 nm.

15. The micellic formulation according to claim 1, wherein the hydrophilic and the lipophilic structural elements are connected via a covalent bond and form a polyethylene glycol-alkyl block copolymer having a molecular weight of 250 to 3,000 g/mole.

16. The micellic formulation according to claim 15, wherein the polyethylene glycol-alkyl block copolymer has a molecular weight of 300 to 1,000 g/mole.

17. The micellic formulation according to claim 1, which has a fluorescence of 797 nm.

18. The micellic formulation according to claim 1, wherein the compound (1) is present in the aqueous media at a 1 weight % concentration.

19. A micellic formulation, comprising 1) a compound (1) comprising polyethylene glycol or methoxypolyethylene glycol as hydrophilic structural element and an alkyl chain as lipophilic structural element, wherein the alkyl chain has 3 to 30 carbon atoms, which are optionally independently mono- or poly-substituted with C.sub.1-C.sub.3 alkyl, hydroxyl or phenyl, and wherein the hydrophilic and the lipophilic structural elements are connected via a covalent bond, which covalent bond is an ether, ester, amid, carbamate, thiocarbamate, thioether, or urea bond, and 2) a near infrared fluorescent dye (2), which is indocyanine green or a derivative of indocyanine green, in which derivative of indocyanine green a) one or two sulfobutyl chains at the indole nitrogen are substituted with —C.sub.1-6-alkyl-R.sup.2, wherein R.sup.2 is selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+—NH.sub.3—N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, —C≡C, —C.sub.1-20-alkyl, —CONH—C.sub.1-20 alkyl, —NHC(O)—C.sub.1-20 alkyl and —O—C.sub.1-20 alkyl, wherein the C.sub.1-20 alkyl is a branched or straight-chain alkyl in which one or more non-consecutive methylene units can be substituted with a unit selected from the group consisting of O, S, NH, C(O)NH, SO.sub.2, SO, aryl, ethene and ethine, and wherein the alkyl is substituted with at least one group selected from the group consisting of —OH, —OSO.sub.3H, —OSO.sub.3.sup.−Na.sup.+, —NH.sub.2, —N.sub.3, —COOH, —SH, —SO.sub.3H, —SO.sub.3.sup.−Na.sup.+, and —C≡C; and/or b) the polymethine chain is substituted with a substituted polymethine chain with a group R.sup.3 at the central carbon atom, wherein the two adjacent carbon atoms can form a 5- or 6-membered ring together with the three carbon atoms of the polymethine chain, wherein R.sup.3 is selected from the group consisting of —C.sub.1-6-alkyl-R.sup.2, -phenyl-C.sub.1-6alkyl-R.sup.2, —S-phenyl-C.sub.1-6alkyl-R.sup.2, —O-phenyl-C.sub.1-6alkyl-R.sup.2, and —O-phenyl-C.sub.1-6alkyl-R.sup.2, wherein R.sup.2 is defined as above; and/or c) the outer benzindole rings are substituted with one or more groups independently selected from the group consisting of —SO.sub.3.sup.−Na.sup.+, —COOH and —OH, wherein micelles have been formed in an aqueous media from the combination consisting of only the compound (1) and the near infrared fluorescent dye (2), wherein the diameter of the micelles is in a range of 1 nm to 50 nm, wherein the fluorescence of the micelles is in a range of 750 to 900 nm, and wherein the micellic formulation contains micelles having a structure wherein the dye (2) is encapsulated by the compound (1).

20. The micellic formulation according to claim 1, which has a 4 times higher fluorescence quantum yield compared to the near infrared fluorescent dye (2) in water.

21. The micellic formulation according claim 1, which has a 4 times higher fluorescence quantum yield compared to the near infrared fluorescent dye (2) in water, wherein the infrared fluorescent dye (2) is indocyanine green.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A: Absorption spectrum of ICG in water (dotted line) and ICG micelles in water after preparation according to Example 1 (solid line). Standardization to micelle solution=1.

(2) FIG. 1B: Fluorescence emission spectrum of ICG in water (dotted line) and ICG micelles in water after preparation according to Example 1 (solid line). Standardization to micelle solution=1.

(3) FIG. 1C: Fluorescence intensity of ICG in water (squares) and of the ICG micelles in water as a function of the concentration of ICG (0.001 mg/mL to 5 mg/mL).

(4) FIG. 1D: Particle size distribution by means of dynamic light-scattering.

(5) FIG. 1E: Determination of stability by measuring the absorption in the maximum of the various formulations as a function of time. Comparison of ICG in water (squares) and three ICG micelle formulations; Solutol 10% (circles), 20% (triangles), 40% (inverted triangles).

(6) FIG. 2A: Absorption spectra of ICG in water and ICG Cithrol 10MS micelles.

(7) FIG. 2B: Emission spectrum of ICG in DMSO and Cithrol 10MS micelles.

(8) FIG. 2C: Stability of ICG (0.005% ICG) in Cithrol 10MS micelles.

(9) FIG. 3A: Absorption spectra of ICG in water and ICG-Crodet S40 LD micelles.

(10) FIG. 3B: Emission spectrum of ICG in DMSO and Crodet S40 LD micelles.

(11) FIG. 3C: Stability of ICG (0.005% ICG) in Crodet S 40 micelles.

(12) FIG. 4A: Absorption spectra of ICG in water and ICG-Brij® 58 micelles.

(13) FIG. 4B: Emission spectrum of ICG in DMSO and Brij® 58 micelles.

(14) FIG. 4C: Stability of ICG (0.005% ICG) in Brij® 58 micelles.

(15) FIG. 5A: Absorption spectra of ICG in water and ICG-Brij® 98 micelles.

(16) FIG. 5B: Emission spectrum of ICG in DMSO and Brij® 98 micelles.

(17) FIG. 5C: Stability of ICG (0.005% ICG) in Brij® 98 micelles.

(18) FIG. 6A: Structure of the ICG derivative NW003.5.

(19) FIG. 6B: Absorption spectra of NW003.5 in water and NW003.5-Solutol HS 15 micelles.

(20) FIG. 6C: Emission spectrum of ICG in DMSO and NW003.5-Solutol HS 15 micelles.

(21) FIG. 6D: Stability of NW003.5 (0.005% NW003.5) in Solutol HS 15 micelles.

(22) FIG. 7A: Structure of the ICG derivative IR-783.

(23) FIG. 7B: Absorption spectra of IR-783 in water and IR-783-Solutol HS 15 micelles.

(24) FIG. 7C: Emission spectrum of ICG in DMSO and IR-783-Solutol HS 15 micelles.

(25) FIG. 7D: Stability of IR-783 (0.005% IR-783) in Solutol HS 15 micelles.

(26) FIG. 8: Schematic view of exemplary dyes for use in the present invention; a) indocyanine green (ICG); b) derivatives of the indocyanine green for use in the present invention.

(27) The invention is described in more detail in the following examples:

EXAMPLES

Example 1

Preparation of the Micelle Formulation

(28) Example 1: 2 g Solutol HS 15 are heated to 65° C. 10 ml water for injection purposes are added under stirring and the clear solution is cooled to room temperature. 50 mg ICG are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(29) Example 2: 2 g Solutol HS 15 are added to 10 ml water for injection purposes under stirring at room temperature. A clear solution is obtained. 50 mg ICG are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(30) Absorption and Fluorescence Measurements

(31) Absorption spectra in a wavelength range of 700 nm to 900 nm were recorded with a UVIKON 933 Spectrophotometer (company Kontron) in the various solvents.
.fwdarw.ICG in water λmax=780 nm
ICO micelles λmax=797 nm  (FIG. 1A)

(32) Fluorescence measurements were carried out by means of a FluoroLog-2 Spectrofluorometer (350 W Xenon lamp) of the company Spex. For this purpose, emission spectra of 700 nm to 900 nm were recorded. The excitation wavelength corresponded to the respective maximum of the formulation in the absorption spectrum (ICG in water λmax=780 nm and ICG micelles λmax=795 nm). Due to the s & r modus of the software DM 3000, the different lamp intensities of the different excitation wavelengths could be taken into account in the evaluation.

(33) The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.13).

(34) .fwdarw. The quantum yield of ICG micelles is Φ=0.08 compared to ICG in water Φ=0.02. Quenching only takes place at higher concentrations (starting at 0.1 mg/ml ICG) (FIG. 1B and FIG. 1C).

(35) Stability Tests

(36) For the stability test the absorption in the maximum of the various formulations was measured as a function of time. For this purpose, 0.0005% ICG solutions were prepared and stored at room temperature. The storage of the purely aqueous ICG formulation shows a reduction of the standardized absorption to below 10% after only 7 days. The micelle formulations of ICG on the other hand still show more than 90% of the absorption after 7 days of storage compared to the initial value, and even after 4 weeks, absorption does not fall below 70% (FIG. 1E).

(37) Particle Size

(38) The particle size distribution was determined by means of dynamic light-scattering (Zetasizer NS, company Malvern). Measurement was carried out with a He—Ne-Laser (633 nm, 4 mW) from an angle of 173°. The samples were measured directly without dilution in 45 μl quartz cuvettes.

(39) .fwdarw. ICG micelles have a hydrodynamic diameter of 12 nm at a PDI (polydispersity index) of 0.061. (FIG. 1D)

(40) Plasma Protein Binding

(41) The wavelength shift in the absorption spectrum was observed to determine the plasma protein binding. For this purpose, spectra of 700 nm to 900 nm of ICG in water and in plasma were compared with spectra of ICG micelles in water and in plasma.

(42) .fwdarw. In both formulations, the absorption maximum in plasma shifts to 805 nm. The behavior of the plasma protein binding of the ICG in the formulations of the present invention corresponds to that of ICG in an aqueous medium.

(43) Hemolysis Assay

(44) For the examination of the hemolytic activity of ICG micelles, heparinized human whole blood was first removed from the plasma and washed 3 times with PBS buffer. After the preparation of a 2% erythrocyte suspension in PBS, it was incubated with the ICG micelle formulation for 1 h at 37° C. Pure PBS solution was used as blank reading value (0% hemolysis) and 2% triton solution was used as 100% hemolysis value. After incubation, the erythrocytes were centrifuged off and the red pigmentation in the supernatant was determined photometrically at 540 nm.

(45) .fwdarw. ICG micelles show no hemolytic activity.

(46) PEG-Alkyl Compounds

Example 2

(47) 0.3 g Cithrol 10 MS (PEG 20 stearat) are dissolved in 10 ml water for injection purposes under stirring at room temperature. A clear solution is obtained. 50 μl of a 1% (w/v) 50 mg ICG solution are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(48) Absorption and Fluorescence Measurements

(49) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectralphotometer in the various solvents.
.fwdarw.ICG in water λmax=779 nm
ICG-Cithrol 10 MS micelles λmax=800 nm  (FIG. 2A)

(50) Fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.12). The quantum yield of ICG-Cithrol 10 MS micelles is φ=0.08 compared to ICG in water at φ=0.02 (FIG. 2B).

(51) Stability Tests

(52) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 2 months of storage at 4° C. and under exclusion of light, the Cithrol 10MS micelle formulations of ICG still showed more than 94% of absorption compared to the initial value (FIG. 2C).

Example 3

(53) 0.4 g Crodet S40 LD (PEG 40 Stearat) are dissolved in 10 ml water under stirring at room temperature for injection purposes. A clear solution is obtained. 50 μl of a 1% (w/v) ICG solution are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(54) Absorption and Fluorescence Measurements

(55) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectralphotometer in the various solvents.
.fwdarw.ICG in water λmax=779 nm
ICG-Crodet S40 LD micelles λmax=800 nm  (FIG. 3A)

(56) Fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.12). .fwdarw. The quantum yield of ICG-Crodet S40 LD micelles is φ=0.07 compared to ICG in water at φ=0.02 (FIG. 3B).

(57) Stability Tests

(58) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 2 months of storage at 4° C. and under exclusion of light, the Crodet S40 LD micelle formulations of ICG still showed more than 97% of absorption compared to the initial value (FIG. 3C).

Example 4

(59) 0.5 g Brij® 58 (PEG 20 cetylether) are dissolved in 10 ml water for injection purposes under stirring at room temperature. A clear solution is obtained. 50 μl of a 1% (w/v) 50 mg ICG solution are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(60) Absorption and Fluorescence Measurements

(61) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectralphotometer in the various solvents.
ICG in water λmax=779 nm
ICG-Brij® 58 micelles λmax=800 nm  (FIG. 4A)

(62) Fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard=0.12). .fwdarw. The quantum yield of ICG-Brij® 58 LD micelles is φ=0.06 compared to ICG in water at φ=0.02 (FIG. 4B).

(63) Stability Tests

(64) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 2 months of storage at 4° C. and under exclusion of light, the Brij® 58 micelle formulations of ICG still showed more than 97% of absorption compared to the initial value.

Example 5

(65) 1 g Brij® 98 (PEG 20 oleylether) are dissolved in 10 ml for injection purposes water under stirring at room temperature. A clear solution is obtained. 50 μl of a 1% (w/v) ICG solution are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(66) Absorption and Fluorescence Measurements

(67) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectralphotometer in the various solvents.
ICG in water λmax=779 nm
ICG-Brij® 98 micelles λmax=800 nm  (FIG. 5A)

(68) Fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.12). .fwdarw. The quantum yield of ICG-Brij® 98 LD micelles is φ=0.06 compared to ICG in water at φ=0.02 (FIG. 5B).

(69) Stability Tests

(70) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 2 months of storage at 4° C. and under exclusion of light, the Brij® 98 micelle formulations of ICG still showed more than 95% of absorption compared to the initial value (FIG. 5C).

(71) ICG-Derivatives

Example 6

(72) 1 g Solutol® HS 15 is dissolved in 10 ml water for injection purposes under stirring at room temperature. A clear solution is obtained. 50 μl of a 1% (w/v) NW003.5 solution (FIG. 6A) are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(73) Absorption and Fluorescence Measurements

(74) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectralphotometer in the various solvents.
.fwdarw.NW003.5 in water λmax (monomer)=788 nm
NW003.5-Solutol® HS 15 micelles λmax=806 nm  (FIG. 6B)

(75) The fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.12). .fwdarw. The quantum yield of NW003.5-Solutol® HS 15 micelles is φ=0.12 compared to ICG in water at φ=0.02 (FIG. 6C).

(76) Stability Tests

(77) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 1 month of storage at 4° C. and under exclusion of light, the Solutol HS 15 micelle formulations of NW003.5 still showed more than 95% of absorption compared to the initial value.

Example 7

(78) 1 g Solutol® HS 15 is dissolved in 10 ml water for injection purposes. Under stirring at room temperature A clear solution is obtained. 50 μl of a 1% (w/v) IR-783 solution (FIG. 7A) are dissolved in the micelle solution and sterilized by filtration through a 0.2 μm membrane filter.

(79) Absorption and Fluorescence Measurements

(80) Absorption spectra were recorded in a wavelength range of 600 nm to 900 nm with a DU®530 Beckman Spectra'photometer in the various solvents.
.fwdarw.IR-783 in water λmax (Monomer)=775 nm
IR-783-Solutol® HS 15 micelles λmax=797 nm  (FIG. 7B)

(81) Fluorescence measurements were carried out by means of a Spectrofluorometer FP-6500 of the company JASCO. For this purpose, emission spectra of 770 to 900 nm were recorded. The excitation wavelength was 760 nm in each case. The quantum yield is calculated via the surface area below the emission curve. ICG in DMSO was used as the standard (Φ=0.12). .fwdarw. The quantum yield of IR-783-Solutol® HS 15 micelles is φ=0.11 compared to ICG in water at φ=0.02 (FIG. 7C).

(82) Stability Tests

(83) For the stability test the absorption spectra of the various formulations were measured as a function of time. After 1 month of storage at 4° C. and under exclusion of light, the Solutol HS 15 micelle formulations of IR-783 showed no significant change in absorption compared to the initial value.