COMPOSITION COMPRISING A FLUOROPHORE LABELLED UPAR-TARGETING PEPTIDE CONJUGATE

Abstract

The present invention describes a composition comprising a fluorophore labelled uPAR-targeting component, a buffer and a surfactant, wherein the fluorophore labelled uPAR-targeting component is solubilized in the composition by means of the surfactant being present, and wherein the composition comprises a maximum of 10 wt % water, preferably a maximum of 5 wt % water.

Claims

1. A composition comprising a fluorophore labelled receptor-targeting component, a buffer and a surfactant, wherein the fluorophore labelled receptor-targeting component is solubilized in the composition by means of the surfactant being present, and wherein the composition comprises a maximum of 10 wt % water, preferably a maximum of 5 wt % water.

2. The composition according to claim 1, wherein the composition comprises a fluorophore labelled receptor-targeting peptide conjugate.

3. The composition according to claim 1, wherein the fluorophore labelled receptor-targeting component is solubilized in the composition at a level corresponding to a single peak with absorption maximum around 800 nm when measuring an absorbance spectrum of the composition in a wavelength area of 700-825 nm.

4. The composition according to claim 1, wherein the fluorophore labelled receptor-targeting component is solubilized in the composition at a level corresponding to having an absorbance spectrum peak with a maximum around 800 nm, and wherein the area of said absorbance spectrum peak is at least 50%, preferably at least 60%, even more preferably at least 65%, of the total area of the absorption spectrum in a given wavelength area of 600-900 nm.

5. The composition according to claim 1, wherein the composition comprises a lyoprotectant, preferably a lyoprotectant chosen from the group of sucrose, trehalose, mannitol, glycine or a combination thereof, more preferably mannitol or mannitol in combination with one or more other components, more preferably a combination of mannitol and glycine or a combination of mannitol and sucrose.

6. The composition according to claim 1, wherein the composition comprises a non-ionic surfactant.

7. The composition according to claim 1, wherein the buffer is provided so that the composition has a physiological pH or substantially a physiological pH, preferably a pH in a range of 7.3-7.5.

8. The composition according to claim 1, wherein the fluorophore labelled receptor-targeting component comprises a fluorophore, a peptide binding to the receptor and a linker group, wherein the fluorophore, the peptide binding to receptor and the linker group is connected by covalent bonds.

9. The composition according to claim 8, wherein the fluorophore is selected from any of indocyanin green (ICG), Methylene blue, 5-ALA, Protoporphyrin IX, IRDye800CW, ZW800-1, Cy5, Cy7, Cy5.5, Cy7.5, IRDye700DX, Alexa fluor 488, Fluorescein isothiocyanate, Flav7, CH1055, Q1, Q4, H1, IR-FEP, IR-BBEP, IR-E1, IR-FGP, or IR-FTAP, preferably wherein the fluorophore is indocyanin green (ICG).

10. The composition according to claim 8, wherein the peptide is chosen from AE101, AE105, AE106, AE110, AE112, AE113, AE116, AE133, AE133*, AE134, AE135, AE136, AE137, AE138, AE139, AE145, AE140, AE141, AE142, AE143, AE144, AE164, AE164*, AE120, AE120* or AE151, wherein the following apply: AE101 is d-Cha-F-s-r-Y-L-W-S, AE105 is D-Cha-F-s-r-Y-L-W-S, AE106 is D-Cha-F-S-r-Y-L-W-S, AE110 is D-Cha-F-s-R-Y-L-W-S, AE112 is D-F-F-s-r-Y-L-W-S, AE113 is D-N-F-s-r-Y-L-W-S, AE116 is D-Cha-F-s-r-G-Y-L-W-S, AE133 is KGSGG-D-Cha-F-s-r-Y-L-W-S, AE133* is KGSGG-D-Cha-F-s-r-Y-L-W-S, AE134 is KGSGG-D-Cha-F-s-r-Y-L-W-A, AE135 is KGSGG-D-Cha-F-s-r-Y-L-A-S, AE is KGSGG-D-Cha-F-s-r-Y-A-W-S, AE137 is KGSGG-D-Cha-F-s-r-A-L-W-S, AE138 is KGSGG-D-Cha-F-s-a-Y-L-W-S, AE139 is KGSGG-D-Cha-F-a-r-Y-L-W-S, AE145 is KGSGG-D-Cha-F-A-r-Y-L-W-S, AE140 is KGSGG-D-Cha-A-s-r-Y-L-W-S, AE141 is KGSGG-D-A-F-s-r-Y-L-W-S, AE142 is KGSGG-A-Cha-F-s-r-Y-L-W-S, AE143 is KGSGG-D-Chp-F-s-r-Y-L-W-S.sup.c, AE144 is KGSGG-D-Cpa-F-s-r-Y-L-W-S.sup.c, AE164 is KGSGG-D-F-F-s-r-Y-L-W-S, AE164* is KGSGG-D-F-F-s-r-Y-L-W-S, AE is [D-Cha-F-s-r-Y-L-W-S]2-/3A-K.sup.c, AE120* is [D-Cha-F-s-r-Y-L-W-S]2-/3A-K.sup.c and AE151 is [r-W-D-Cha-S-L-s-F-Y]2-/3A--K.sup.c, or a combination thereof, or a peptide with a sequence homology of at least 80% to any of these peptides.

11. The composition according to claim 1, wherein the receptor is uPAR.

12. The composition according to claim 1, wherein the fluorophore labelled receptor-targeting component is ICG-Glu-Glu-AE105: ##STR00004## or a pharmaceutically acceptable salt thereof.

13. The composition according to claim 12, wherein the concentration of ICG-Glu-Glu-AE105 is in the range of from 0.1 -10 mg/ml.

14. The composition according to claim 1, wherein the fluorophore labelled receptor-targeting component is ICG-Glu-Glu-AE105: ##STR00005## or a pharmaceutically acceptable salt thereof, wherein the concentration of ICG-Glu-Glu-AE105 is in the range of from 0.1-10.0 mg/ml, wherein the composition also comprises a buffer in the form of sodium phosphate in concentration of 5-50 mM, and wherein the composition comprises a cryoprotectant combination of mannitol in a concentration of 10-50 mg/ml and glycine in a concentration of 1-30 mg/ml.

15. The composition according to claim 1, wherein the composition comprises a polysorbate, preferably Polysorbate, more preferably Polysorbate 20 in a concentration of >0.01 wt %.

16-27 (canceled)

28. A lyophilized composition comprising the composition according to claim 1.

29. A method for the production of a composition according to claim 1, wherein said method comprises admixing the fluorophore labelled receptor-targeting component, the buffer, and the surfactant to solubilize the fluorophore labelled receptor-targeting component in the composition.

30. The method according to claim 29, wherein a lyoprotectant is admixed with the composition so that a lyo cake is produced.

31. The method according to claim 29, wherein said method comprises (i) mixing ICG-Glu-Glu-AE105: ##STR00006## or a pharmaceutically acceptable salt thereof, with sodium phosphate, mannitol and glycine to yield a composition comprising ICG-Glu-Glu-AE105 in a concentration of 0.1-10.0 mg/ml; sodium phosphate in a concentration range of 5-50 mM; mannitol in a concentration range of 10-50 mg/ml; glycine in a concentration range of 1-30 mg/ml; Polysorbate 20 in a concentration of >0.01 wt %. (ii) adjusting the pH of the composition of step (i) to a pH in a range of 6.9-7.9; (iii) transferring amounts of the mixture from step (ii) equivalent to the desired dosage into a suitable container; (iv) drying the mixture; and (v) sealing the container.

32. An optical imaging method comprising the steps of: (a) administering of a composition according to claim 1 accumulating in a target tissue, (b) illuminating the target tissue with light of a wavelength absorbable by the fluorophore; and (c) detecting fluorescence emitted by the fluorophore and forming an optical image of the target tissue.

Description

EXAMPLES

Example 1 Development of an Aqueous Formulation of a Composition According to the Present Invention

[0080] A composition comprising the fluorophore labelled receptor-targeting peptide conjugate ICG-Glu-Glu-AE105 (called composition 1 below) is soluble in DMSO in concentrations up to 20 mg/ml with a peak spectral absorption at 800 nm similar to indocyanine green (ICG) currently used in the clinic for visual assessment of vessels, blood flow and related tissue perfusion, fig. A.

[0081] An aqueous formulation of composition 1 of 1.0 mg/ml has been developed according to the present invention, using excipients suitable for pharmaceutical formulation development.

[0082] Composition 1 is not soluble in phosphate buffer saline at the tested concentration of 1.0 mg/ml, which can be observed both from the spectral absorption in the range from 700 nm to 825 nm with a clear double peak and the corresponding emission spectrum with a significantly lower fluorescent peak at 800 nm, fig. A. Excitation was conducted at 775 nm.

[0083] In a 10 mM sodium phosphate buffer containing 45 mg/ml mannitol, pH 7.4 the peak absorption shifted towards the peak absorption at 800 nm and in a formulation containing 10 mM sodium phosphate, 45 mg/ml mannitol, 1% Polysorbate 20, pH 7.4 the absorption spectrum was similar to the spectrum obtained in DMSO. Addition of 0.2 g/ml of 2-Hydroxypropyl-β-cyclodextrin (HBC) to phosphate buffer saline was also very efficient in solubilizing composition 1 in 1.0 mg/ml, fig. B.

[0084] The combined absorbance and emission spectra for the three formulations (1.0 mg/ml) with complete solubilization of composition 1 are presented in figs. C and D.

Further Explanation of the Figs. A-D
A. Absorbance and emission spectrum of FG001 dissolved in DMSO and PBS at 1.0 mg/ml. Excitation wavelength: 775 nm. Analytical samples for absorbance measurement were further diluted 200-fold and 400-1000-fold for emission spectra.
B. Absorbance spectrum of FG001 dissolved in 1) 10 mM sodium phosphate, 45 mg/ml mannitol, pH 7.4, 2) 10 mM sodium phosphate, 45 mg/ml mannitol, 1% Polysorbate 20, pH 7.4 and 3) PBS, 0.2 g/ml HBC. Analytical samples for absorbance measurement were further diluted 200-fold and 400-1000-fold for emission spectra
C. Overlay of absorption spectra for the three formulations (1.0 mg/ml) with complete solubilization of FG001: A) DMSO, B) 10 mM sodium phosphate, 45 mg/ml mannitol, 1% Polysorbate 20, pH 7.4 and C) phosphate buffer saline, 0.2 g/ml HBC. Analytical samples for absorbance measurement was further diluted 200-fold and 400-1000-fold for emission spectra
D. Overlay of emission spectra for the three formulations (1.0 mg/ml) with complete solubilization of composition 1: A) DMSO, B) 10 mM sodium phosphate, 45 mg/ml mannitol, 1% Polysorbate 20, pH 7.4 and C) phosphate buffer saline, 0.2 g/ml HBC. Excitation wavelength: 775 nm. Analytical samples for absorbance measurement were further diluted 200-fold and 400-1000-fold for emission spectra

Example 2 Determination of the Optimal Concentration Range of Polysorbate 20 for Solubilization of Composition 1—See Fig. E

[0085] The optimal concentration range of Polysorbate 20 for solubilization of composition 1 at a concentration of 1.0 mg/ml in 10 mM sodium phosphate, 45 mg/ml mannitol, pH 7.4 was evaluated by measuring the absorption spectrum for formulations containing 0 to 1.0% Polysorbate 20.

[0086] The absorptions spectra showed that all formulations containing >0.01% Polysorbate 20, composition 1 was fully solubilized with the main peak spectral absorption at 800 nm.

Example 3 Determination of the Stability of Composition 1 in Three Formulations with Polysorbate 20

[0087] The stability of composition 1 at a concentration of 1.0 mg/ml was evaluated in three different formulations suitable for development of a lyophilized product.

A (Mannitol):

[0088] 10 mM sodium phosphate, 45 mg/ml mannitol, 0.025% Polysorbate 20, pH 7.4

B (Mannitol/glycine):

[0089] 10 mM sodium phosphate, 26 mg/ml mannitol, 8.7 mg/ml glycine, 0.025% Polysorbate 20, pH 7.4

C (Mannitol/sucrose):

[0090] 10 mM sodium phosphate, 26 mg/ml mannitol, 40 mg/ml sucrose, 0.025%

Polysorbate 20, pH 7.4

[0091] Composition 1 was weighed into the formulation buffer and pH was adjusted to 7.4 using sodium hydroxide or hydrochloric acid. The bulk formulation was sterile filtered through suitable sterile filters and filled into 6R vials. The vials were placed with stoppers in a lyophilizer and lyophilized using a standard program. Following lyophilization all vials were capped.

[0092] One vial of each formulation was reconstituted with water for injection prior to measuring osmolality and purity using RP-HPLC as described in example 4.

[0093] Osmolality and purity of composition 1 formulations following reconstitution

TABLE-US-00001 Purity, main peak Extra peak Osmolality (area % (area % Formulation (mOsmol/kg) 780 nm) 780 nm) A: Mannitol 289 94.3 1.1 B: Mannitol/glycine 283 95.4 <0.1 C: Mannitol/sucrose 307 93.9 1.4

[0094] All three formulation had the desired osmolality for a pharmaceutical formulation to be used for intravenous administration to humans.

[0095] The stability of the reconstituted, liquid formulations of composition 1 was evaluated at room temperature and daylight (RTL) for two weeks.

TABLE-US-00002 Purity, main peak Extra peak Time (area % (area % Formulation (days) 780 nm) 780 nm) A: Mannitol 0 94.3 1.1 7 93.5 1.9 14 93.0 2.3 B: Mannitol/ 0 95.4 <0.1 glycine 7 95.2 0.2 14 95.1 0.1 C: Mannitol/ 0 93.9 1.4 sucrose 7 92.7 2.7 14 92.7 2.6

[0096] It is well known that fluorophores like ICG are light sensitive and should be protected from light. However, to select the most stable formulation, the three liquid formulations were exposed to day light for two weeks at room temperature.

[0097] From the data presented in the table above, it is evident that formulation B: Mannitol/glycine is the most stable.

[0098] The stability of lyophilized product from the three formulations was also evaluated for two weeks at three different storage conditions:

I: Room Temperature, Daylight (RTL)

II: Room Temperature, Dark (RTD)

III: 40° C., Dark (40D)

[0099]

TABLE-US-00003 Purity, main peak Extra peak Storage (area % (area % Formulation condition 780 nm) 780 nm) A: Mannitol RTL 95.0 0.3 RTD NA* NA 40D 94.6 0.3 B: Mannitol/ RTL 94.8 0.1 glycine RTD 95.2 0.1 40D 94.8 0.1 C: Mannitol/ RTL 94.7 0.3 sucrose RTD 95.1 0.3 40D 94.7 0.2 *NA: Not sufficient lyophilized vials of formulation A

[0100] The stability of the lyophilized vials was significantly increased compared to the liquid formulation and smaller changes were observed. The study confirmed that Formulation B: Mannitol/glycine as lyophilizate has excellent stability both at elevated temperatures (40° C.) and upon exposure to light at room temperature.

[0101] The lyophilized samples stored at Room Temperature, Dark (RTD) and 40° C., Dark (40D) for two weeks were reconstituted and stored for an additional 24 hours at Room Temperature, Daylight (RTL).

TABLE-US-00004 Purity, main peak Extra peak Storage (area % (area % Formulation condition 780 nm) 780 nm) A: Mannitol 2 weeks lyo at RTD, NA* NA 24 hours liquid at RTL 2 weeks lyo at 40D, 94.1 0.7 24 hours liquid at RTL B: Mannitol/ 2 weeks lyo at RTD, 94.7 0.1 glycine 24 hours liquid at RTL 2 weeks lyo at 40D, 94.7 0.1 24 hours liquid at RTL C: Mannitol/ 2 weeks lyo at RTD, 93.9 0.9 sucrose 24 hours liquid at RTL 2 weeks lyo at 40D, 93.9 0.8 24 hours liquid at RTL *NA: Not sufficient lyophilized vials of formulation A

[0102] The stability of samples kept as a lyophilized product for two weeks and following reconstitution stored at room temperature, daylight for 24 hours also supports the selection of Formulation B: Mannitol/glycine as the preferred formulation.

[0103] It should be noted that another possible method to use to investigate the characteristic single absorbance spectrum peak is to calculate the single peak area positioned around 800 nm and to compare this with the total area of the absorption spectrum in a given wavelength area of 600-900 nm. This is further explained above in the description.

Example 4 Determination of Long Term Storage Stability of Composition 1 in One Formulation with Polysorbate 20

[0104] The long term stability of composition 1 at a concentration of 1.0 mg/ml was evaluated as a lyophilizate in one formulation comprising 10 mM sodium phosphate, 26 mg/ml mannitol, 9.0 mg/ml glycine, 0.025% Polysorbate 20, pH 7.4 Composition 1 was weighed into the formulation buffer and pH was adjusted to 7.4 using sodium hydroxide or hydrochloric acid. The bulk formulation was sterile filtered through suitable sterile filters and filled into 6R vials. The vials were placed with stoppers in a lyophilizer and lyophilized using a standard program. Following lyophilization all vials were capped, visual inspected and stored protected from light at 5° C., 25° C./60% RH or 40° C./75% RH for up to 9 months

[0105] At the selected timepoint, a vial is removed from the stability chamber for visual inspection of the lyophilizate and water content determination. Another vial is reconstituted with water for injection prior to analyzing the corresponding liquid formulation for purity, pH and visual appearance.

[0106] Purity of reconstituted composition 1 formulations following storage at 5° C., 25° C./60% RH or 40° C./75% RH for up to 9 months

TABLE-US-00005 Purity, main peak Purity, main peak Purity, main peak (area % 780 nm) (area % 780 nm) (area % 780 nm) Time following storage following storage following storage (months) at 5° C. at 25° C./60% RH at 40° C./75% RH 0 96.6 96.6 96.6 1 96.2 96.2 96.0 3 96.5 96.6 95.9 6 96.3 95.6 93.9 9 96.2 96.2 Not analyzed

[0107] Residual water, visual inspection before/after reconstitution and pH of composition 1 formulations following storage at 5° C. or 25° C./60% RH for up to 9 months.

TABLE-US-00006 Residual water, visual Residual water, visual inspection and pH following inspection and pH following storage at 5° C. storage at 25° C./60% RH Visual Visual inspection inspection Residual before/after Residual before/after Time water recon- water recon- (months) (%) stitution pH (%) stitution pH 0 1.0 Green 7.4 1.0 Green 7.4 1 1.2 solid/clear 7.3 1.8 solid/clear 7.3 3 1.8 green 7.4 1.7 green 7.4 6 1.7 solution 7.3 1.7 solution 7.3 9 1.6 7.4 1.9 7.4

[0108] The selected formulation of composition 1 is shown to be stable for up to 9 months at storage conditions 5° C. and 25° C./60% RH judged by the purity determination. pH of the formulation as well as the appearance of the lyophilizate and the clarity of reconstituted liquid formulation remains unchaged for up to 9 months storage. Furthermore, the residual water content is around 2% following 9 months storage at 25° C./60% RH.

Analytical Methods:

[0109] RP-HPLC to detect purity of reconstituted composition 1: Mobile phase A was composed of purified water/acetonitrile (80/20 v/v) with 5 mM ammonium acetate and mobile phase B was composed of purified water/acetonitrile (10/90 v/v) with 5 mM ammonium acetate. A Waters XBridge BEH Peptide, 3.5 μm, 130Å, 4.6×150 mm column was used. Flow rate was set to 1.0 mL/min,detection was at a wavelength of 780 nm, the column running temperature was 45° C. The sample cooler temperature was set at 5° C. and the sample injection load was 10 μg.

[0110] The water content of the lyophilizate was determined by Karl Fisher titration according to Ph. Eur. 2.5.32.