OPTICAL IMAGING AGENTS TARGETING INFLAMMATION

Abstract

##STR00001##

The present invention relates to an optical imaging agent of formula (SIGNAL).sub.n-SUPPORT-(L-BIOVECTOR).sub.m, wherein: SUPPORT represents a physiologically acceptable chemical or biological substrate, with a particle size of between 1 and 100 nm, SIGNAL is a fluorophore, L is a linker of formula C(X)R.sup.1Y, with X being O, NH or S R.sup.1 being a (C.sub.1-C.sub.6)alkyl group, optionally R.sup.1 being a (C.sub.1-C.sub.6)alkyl group, optionally interrupted by 1 to 3 groups selected from O, NH, C(O), NHC(O), (O)CNH, C(O)NHNC, NC, and (I), Y being NH or a (O)CNH group, and BIOVECTOR is a carbohydrate targeting markers of inflammation, advantageously selected from the group consisting of mannose, sialyl Lewis.sup.X and derivatives thereof, n is greater than or equal to 0.5 and is less than 2, m is between 0 and 30, preferably between 1 and 30, more preferably between 5 and 20, diagnostic compositions comprising same and use thereof as a diagnostic agent (in vivo or ex vivo), or as contrast agent for image-guided surgery.

Claims

1. Optical imaging agent of formula (SIGNAL).sub.n-SUPPORT-(L-BIOVECTOR).sub.m, wherein: SUPPORT represents a physiologically acceptable chemical or biological substrate, with a particle size of between 1 and 100 nm, SIGNAL is a fluorophore, L is a linker of formula C(X)R.sup.1Y, with X being O, NH or S, R.sup.1 being a (C.sub.1-C.sub.6)alkyl group, preferably a (C.sub.3-C.sub.5)alkyl group, optionally interrupted by 1 to 3 groups selected from O, NH, C(O), NHC(O), (O)CNH, C(O)NHNC, NC, and ##STR00042## Y being NH or (O)CNH, and BIOVECTOR is a carbohydrate able to target markers of inflammation, n is greater than or equal to 0.5 and is less than 2, m is between 1 and 30, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

2. The optical imaging agent of claim 1, wherein SUPPORT is a protein.

3. The optical imaging agent of claim 1, wherein SUPPORT is an albumin with a particle size of between 1 and 100 nm.

4. The optical imaging agent of claim 1, wherein SIGNAL is of formula (I): ##STR00043## wherein q is 0 or 1, r is 0 or 1, R.sub.b1 and R.sub.b2 are H or a C.sub.1-C.sub.4 group, or are bridged to form a CH.sub.2CH.sub.2CH.sub.2 alkylene group (only when q and r represent 1), R.sub.a1 and R.sub.a2 are identical or different and are independently a (C.sub.1-C.sub.6)alkyl group, optionally substituted by a SO.sub.3.sup. group, a SO.sub.3K group, a SO.sub.3Na group, or a COOH group, provided that not more than one of R.sub.a1 and R.sub.a2 is substituted by a SO.sub.3.sup. group, Ring A represents a C.sub.5-C.sub.6 monocyclic aryl or heteroaryl group or a C.sub.8-C.sub.12 fused bicyclic aryl or heteroaryl group, provided that the compound of formula (I) comprises at least one SO.sub.3.sup. or SO.sub.3Na group, but not more than one SO.sub.3.sup. group, and that when the compound of formula (I) has an overall positive electric charge, it is provided as a salt, in particular a halogenide salt such as a chloride Cl.sup. salt.

5. The optical imaging agent of claim 1, wherein X is NH and R.sup.1 is a (C.sub.1-C.sub.6) alkyl, optionally interrupted by a ##STR00044## group.

6. The optical imaging agent of claim 1, wherein BIOVECTOR is of formula (II): ##STR00045## wherein R.sub.2 represents a (C.sub.1-C.sub.6)alkyl group, and X represents a heteroatom such as O, S or NH, or a compound of formula (III) or (IV) ##STR00046## wherein R is R.sub.2X, with R.sub.2 being a (C.sub.1-C.sub.6)alkyl group and X being a heteroatom such as O, S or NH, and Rs is H, SO.sub.3K or SO.sub.3Na.

7. The optical imaging agent of claim 1, wherein BIOVECTOR is of formula (V), (VI) or (VII): ##STR00047## wherein R is R.sub.2X, with R.sub.2 being a (C.sub.1-C.sub.6)alkyl group and X being a heteroatom such as O, S or NH.

8. A diagnostic composition comprising at least one optical imaging agent according to claim 1, a pharmaceutically acceptable salt, solvate or hydrate thereof, and at least one pharmaceutically acceptable excipient.

9. An in vivo diagnostic method comprising administering to a patient in need thereof an effective amount of the optical imaging agent according to claim 1.

10. The in vivo diagnostic method according to claim 9, comprising diagnosing diseases or conditions associated with inflammation.

11. The in vivo diagnostic method according to claim 10, wherein the disease or condition associated with inflammation is selected from stroke, renal failure or cancer.

12. The in vivo diagnostic method according to claim 9, wherein said method is applied to surgical resection of tumours in the patient and comprises the following further successive steps: resecting the tumoral tissue identified prior to surgery in an area of interest; imaging the area of interest of the patient, to whom the effective amount of the optical imaging agent has been administered prior to surgery or during surgery, using an optical imaging device, so as to identify remaining tumoral tissues; when light signals are observed in the area of interest defining remaining tumoral tissues, proceeding with the resection of the identified remaining tumoral tissues; when no light signal is observed in the area of interest, not proceeding with any further resection in the area of interest.

13. A method of imaging a biological tissue ex vivo, wherein said biological tissue comprises the optical imaging agent according to claim 1, the method comprising applying optical or fluorescence imaging to said tissue.

14. The method of claim 13, wherein the biological tissue is a biopsy sample.

15. The method of claim 13, further comprising a step of diagnosing a disease or condition associated with inflammation from said optical or fluorescence imaging.

16. The optical imaging agent of claim 1, wherein SUPPORT is a human serum albumin with a particle size of between 1 and 100 nm.

17. The optical imaging agent of claim 4, wherein Ring A represents a C.sub.5-C.sub.6 monocyclic aryl group or a C.sub.8-C.sub.12 fused bicyclic aryl group, such as a phenyl or naphthyl group.

18. The optical imaging agent of claim 4, wherein Ring A is substituted by one SO.sub.3.sup. or SO.sub.3Na group, provided that not more than one of R.sub.a1 and R.sub.a2 is substituted by a SO.sub.3.sup. group.

19. The optical imaging agent of claim 6, wherein R.sub.2 represents a (C.sub.1-C.sub.4)alkyl group and X represents NH.

Description

FIGURES

[0207] FIG. 1: A: Mass spectrum of the imaging agent and B: Mass spectrum of albumin (M=90582) as a reference (M=66330). These two spectra show the full conversion of albumin into the functionalized albumin of Example 1.

[0208] FIG. 2: Mass spectrum of the cyanin-5-Albumine-mannose imaging agent of Example 2 (HSA-Mannose).

[0209] FIG. 3: Fluorescence intensity of the albumin reacted with 2 equivalents or 20 equivalents of cyanin. FIG. 3 shows that less equivalents provide a higher fluorescent intensity.

[0210] FIG. 4: Cell Expression of E selectin. Tumor cells are CT26, Endothelial cells are Bend3 (A). In vitro evaluation of the binding (B-C) and the internalization (D) of HSA and HSA-Slx (SLX) in endothelial and tumor cells.

[0211] FIG. 5: Image-guided surgery: images A and B show that imaging under fluorescence (B and C) provides a higher sensitivity to image the tumoral area than white light (1). The Tumor can be resected under normal light (D), the fluorescence is put on again to evaluate residual tumor margin remaining post-surgery (E). The tumor on the side of the animal is also fluorescent.

[0212] FIG. 6: Biodistribution of HSA-Slx in BALB/c mice with subcutaneous colorectal CT 26 tumor.

[0213] FIG. 7: Accumulation of the fluorescent albumin-sialyl lewis X derivative (HSA-Slx) in CT26 colon tumors 24 hours post-injection observed by optical imaging. BG=background, Control=Albumine without biovector (HSA-CY5), sialyl refers to the optical imaging agent of Example 1 (HSA-Slx).

[0214] FIG. 8: Accumulation of HSA-Slx in DBA mice collagen induced arthritis model

[0215] FIG. 9: Distribution of HSA, HSA-Lactose (HSA-L-CYS) and HSA-Mannose (HSA-M-CY5) in the liver of balbc mice as function of time after intravenous injection.

[0216] FIG. 10: Distribution into monomers, dimers, trimers or oligomers of native albumin (Clinical HSA), cyanine-grafted albumin (HSA-Cyanine) and optical imaging agents of the present invention HSA-SLX and HSA-SLX2.

[0217] FIG. 11: Ex vivo imaging of extracted organs 24 h after injection of HSA grafted with the tetrasaccharide Sialyl Lewix X (HSA-SLX) or HSA grafted with the trisaccharide analog of the Sialyl Lewis X of the invention described above (HSA-SLX2).

[0218] FIG. 12: Quantification of the ex vivo images with M3vision software given as the percentage of fluorescence per mice in the organs for HSA grafted with the tetrasaccharide Sialyl Lewis X (HSA-SLX) or HSA grafted with the trisaccharide analog of the sialyl Lewis X of the invention (HSA-SLX2).

EXAMPLES

[0219] The following examples are given for illustrative purpose only, and shall not be construed as limiting in any way.

[0220] In the following PBS stands for Phosphate Buffered Saline solution (as known in the art), and EDTA stands for ethylenediaminetetraacetic acid.

Example 1

Synthesis of an Optical Imaging Agent According to the Invention Using a Derivative of SLe.SUP.X .as BIOVECTOR (Compound HSA-Slx)

[0221] Albumin (2 mg) is coupled to N-N-disulfonate-cyanin-5-N-hydroxysuccinimide:

##STR00037##

available from luminoprobe (2 equivalents per amine) in PBS and left for 30 minutes under gentle stirring. The mixture is washed with an ultrafiltration device with a 50 kDa cut off at 2000 rpm during 30 minutes at 4 C. Sialyl lewis-X-maleimide is then coupled to the labelled albumin in PBS/EDTA at room temperature during 30 minutes in presence of 2-iminothiolane hydrochloride (10 equivalent per amine). A washing step is performed to remove the excess of iminothiolane and obtain the labelled targeting albumin HSA-Slx in NaCl 0.9%. The full functionalisation of albumin was confirmed by mass spectrometry with a mass of 90782 for the imaging agent (FIG. 1).

[0222] Sialyl lewis-X-maleimide is obtained as follows. To Sialyl Lewis X (described in Lu et al. Carbohydrate research 383(2014) 89-96) is added of N-Succinimidyl 3-maleimidopropionate (hereafter NHS-maleimide):

##STR00038##

and triethylamine at room temperature, the reaction mixture is stirred for 2 hours at room temperature. The reaction mixture is then evaporated and the crude product is purified using column chromatography on silica gel.

Example 2

Synthesis of an Optical Imaging Agent According to the Invention Using a Derivative of Mannose as BIOVECTOR (Compound HSA-Mannose)

Step 1:

[0223] Albumin (20 mg) is coupled to N-N-disulfonate-cyanin-5-N-hydroxysuccinimide (0.1 mg) in 1 mL PBS and left for 30 minutes under gentle stirring. The mixture is washed with an ultrafiltration device with a 50 kDa cut off at 2000 rpm during 30 minutes at 4 C. The intermediate compound is the cyanine-labelled albumin, which does not contain any targeting portion (no BIOVECTOR). This intermediate cyanine-labelled albumin is referred to hereinafter as HSA-CY5.

Step 2:

[0224] Mannose-maleimide is then coupled to the labelled albumin HSACY5 in PBS/EDTA at room temperature during 30 minutes in presence of 2-iminothiolane hydrochloride (10 equivalent per amine). A washing step is performed to remove the excess of iminothiolane and obtain the labelled targeting albumin in NaCl 0.9%. The expected mass of the imaging agent was confirmed by mass spectrometry (M 79636) (FIG. 2). The reaction of 2 equivalents of cyanin gave the highest intensity of fluorescence as referred to 20 equivalents (FIG. 3).

Mannose-maleimide

[0225] ##STR00039##

is obtained as follows. 2-aminoethylmannopyranoside

##STR00040##

is dissolved in DMF. NHS-maleimide (491 mg, 2.2 mmol) is added, and the reaction mixture is stirred overnight. The reaction mixture is then concentrated in vacuo, and the crude product is purified using column chromatography on silica gel (eluent:dichloromethane/MeOH:8/2). 339 mg of purified Mannose-maleimide is obtained.

Example 3

In Vitro Binding Affinity of the Optical Imaging Agent of Example 1

[0226] The in vitro experiments were performed with bEnd.3 mus musculus brain endothelial cells (CRL-2299) and CT26.WT mus musculus colon carcinoma cells (CRL-2638) provided by ATCC. Those cells were cultured in DMEM-Dulbecco's Modified Eagle Medium (10566-016, Gibco Thermo Fisher) added by 10% of fetal bovine serum (10500056, Gibco Thermo Fisher) and 1% of Penicillin-Streptomycin (5,000 U/mL, 15070063, Gibco Thermo Fisher) antibiotics. A suspension of Lipopolysaccharide stimulated cells (0.1 mg/mL, 4 h, 37 C.), were collected and prepared in binding buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl2, 1% BSA in water) at 500 000 cell per milliliter. An incubation of 1 h at 4 C. was realized in order to saturate unspecific binding. For evaluation of E-selectin expression (FIG. 4A) cells were incubated successively with purified rat anti-Mouse CD62E antibody (1 g/mL, 550290 BD Pharmingen) in PBS/BSA 1% and with goat anti-rat IgG-Alexa Fluor488 (1 g/ml, ab150157 Abcam) for 30 min a 4 C. To study cellular binding and internalization, cells were incubated with 30 ng of HSA or HSA-SLX for 20min at 4 C. (FIG. 4B-C) or 37 C. (FIG. 4D).

[0227] After those different steps, cells were washed twice with binding buffer and analyzed by flow cytometry (Guava easyCyte Millipore).

[0228] We can see that both tumor cells and endothelial cells express E-selectin. The imaging agent bind to both type of cells and is internalized mostly by endothelial cells which express E-selectin at a higher level. The non-functionalized albumin (control) neither bind, nor is internalized by endothelial cells or tumor cells.

Example 4

Demonstration of the Efficacy of the Optical Imaging Agent of Example 1 as an Aid to Surgery

[0229] BalbC female mice (from janvier labs) were implanted with 3 mm.sup.2 of CT26 tumour fragment in subcutaneous way. Fifteen day after implantation, mice were anesthetized with a mixture of ketamine (80 mg/kg, Clorketam 1 000 Vetoquinol) and xylazine (10 mg/kg, ROMPUN 2% Bayer), 200 l of HSA-SLX-Cy5 (2.7 mg/ml) suspension was then injected into the tail vein. Twenty four hour after injection, an evaluation of image guided surgery (FIG. 5) was realized under mice anaesthesia with Fluobeam open Imaging system for in vivo near infrared fluorescence imaging ( ex 690, em>700 nm).

Example 5

In Vivo Liver Accumulation of the Optical Imaging Agent of Example 1

[0230] Female balbc mice (from janvier labs) were anesthetized with ketamine/xylazine mixture, 5 mice per condition were injected with HSA-CY5 and HSA-Slx suspension (2.7 mg/ml) in the tail vein of Balbc mice 14 days after CT26 tumour implantation. The biodistribution kinetic of formulation was recorded by Fluobeam system as function of time (FIG. 6). Twenty four hours after HSA-CY5 and HSA-SLX-CY5 injection, ex vivo quantification of the signal accumulated in the tumour was evaluated after blood elimination by mice PBS perfusion. Those results (FIG. 7) were imaged and quantified by Photon IMAGER optima (Biospace lab) and finally expressed in photon per second per steradian (ph/s/sr). Of note, HSA-CY5, which does not contain any targeting portion (BIOVECTOR) is used as a control.

Example 6

In Vivo Paw Accumulation of the Optical Imaging Agent in Collagen-Induced Arthritis Model of Example 1

[0231] Collagen Arthritis model was induced in DBA/1 mice (from janvier labs) by injection with bovine type II collagen emulsified in Complete Freund's Adjuvant in the posterior paw articulation. Twenty four hours after arthritis induction, mice were anesthetized and injected with HSA-SLX-CY5, suspension (2.7 mg/ml) in the tail vein. In vivo kinetic of the formulation was recorded by Fluobeam system at different time points (FIG. 8A). Twenty for hours after probe injection colocalization signal between probes and arthritis articulation could be evaluated by PhotonlMAGER optima (Biospace lab) after luminol injection (FIG. 8B).

Example 7

Biodistribution and Ex Vivo Quantification of the Fluorescence as Regard to the Agent without Bio Vector in Healthy Mice

[0232] Female balbc mice (from janvier labs) were anesthetized with ketamine/xylazine mixture, mice were injected with HSA-CY5, HSA-Lactose (HSA-L-CY5, obtained as described in example 1, but substituting lactose for mannose) or HSA-Mannose (HSA-M-CY5) suspension (2.7 mg/ml) in the tail vein of Balbc healthy mice. In vivo liver kinetic of each formulation was recorded and quantified by the use of PhotoniMAGER optima (Biospace lab). The percent of the fluorescence detected in the liver was calculated as function of the signal of the entire mice (FIG. 9).

Example 8

Oligomers and Aggregates Distributions of Native Albumin, Cyanine Grafted Albumin and Optical Imaging Agents According to the Invention HSA-SLX and HSA-SLX2

[0233] Size exclusion chromatography/UV was performed to evaluate the distribution of monomers, dimers, trimers and oligomers of the optical imaging agents according to the present invention.

Experimental:

[0234] Separation and analysis were performed on a LC-10 liquid chromatography system from Shimadzu (Kyoto, Japan) natively equipped with a vacuum degasser, an auto-sampler, a UV absorbance detector and refractive index detector (RID). The size exclusion chromatography stationary phase was a Shodex Protein LW 803 column provided by Showa Denko (Japan). Between the auto-sampler and HPLC pump, mobile phase was filtered in-line by a 0.1 m durapore PVDF membrane in a PEEK in-line filter. Detection was realized using UV absorbance at a wavelength of 280 nm. Concomitantly, refractive index quantifications were performed using white light. Mobile phase was CH3COONH4 50 mM NaN3 0.03% in milli-Q water degassed by vacuum pump and filtered on stericup filter units. MALS detection was achieved on a three angle mini-Dawn Treos II from Wyatt Technology equipped with a 658 nm wavelength laser. Data analysis was achieved on Astra 7.1, Wyatt Technology.

[0235] HSA-SLX corresponds to the optical imaging agent described in example 1.

[0236] HSA-SLX2 corresponds to the optical imaging agent described in example 1, except that the SLX moiety has been replaced by the trisaccharide of the invention with the formula below and whose synthesis is described in the above Process of preparation section:

##STR00041##

wherein Rs is SO.sub.3Na.

Results are Illustrated on FIG. 10.

[0237] The optical imaging agents according to the present invention, HSA-SLX and HSA SLX2, exist mainly in the form of monomers and form very few oligomers. Optical imaging agents according to the invention therefore barely form aggregates.

Example 9

Biodistribution of the Probe by Optical Imaging

[0238] Female BALB/cJRj 8 weeks old mice (Janvier labs, Le Genest-Saint-Isle, France) were anesthetized with a mixture of 100 mg kg1 of ketamine (IMALGENE 1000 Boehringer Ingelheim, France) and 10 mg kg1 of xylazine (Rompun, Bayer, France) and injected by an intravenous route with 200 l of HSA-SLX or HSA-SLX2 solutions, with both concentrations at 1 mg mL1. Twenty four hours after the injection, the mice were sacrificed, their PBS perfused organs were placed under the camera to acquire the fluorescence signal associated with the organs (5 s, ex 640 nm, em 780 nm). (FIG. 11)

[0239] The images were processed using the M3vision software. The result is expressed as percent of the fluorescence found per mice (FIG. 12).

HSA SLX and HSA SLX 2 are as Described in Example 8.

[0240] Sialyl Lewis X is known in the art as the best agent to target selectin E, due to its high affinity for its target. The trisaccharide analog SLX2 of the invention appears to have the same affinity for the target than SLX and is easier to synthesize. Moreover, as shown here, grafting this analog to albumin confers similar property to the in vivo imaging agent, as a similar signal of fluorescence is observed with either HSA grafted with SLX or SLX2.