Patent Description:
Recently, optical imaging has drawn increasing interest because of the ease of use of portable imaging devices, and reduced toxicity and risk to the operator and patient compared in particular with X-ray imaging, radiomedicine or magnetic resonance imaging.

Optical imaging, and more specifically fluorescence imaging, are used for analysis of biological samples (such as biopsies for instance, hereafter referred to as "ex vivo imaging"), in <NUM> dimensions (2D) as well as <NUM> dimensions (3D) (for instance using tomography), and can be used in vivo during surgery, for instance using portable devices such as those commercialized by Fluoptics and Novadaq.

Optical imaging requires administering optical agents to the patient or animal to be imaged (in vivo or ex vivo) prior to imaging. For instance, <NPL>, (<NUM>. <NUM>)) describes an optical imaging probe, comprising: a glycol chitosan nanoparticle with attached cyanine fluorophore and maleimide-PEG-mannose targeting inflammation.

However, to improve the quality and accuracy of diagnostic using optical imaging, there remains a need for optical imaging agents able to target specific markers in the body. In particular, there is a need for optical imaging agents selectively targeting markers of inflammation, in order to diagnose inflammatory diseases in vitro or ex vivo or in vivo. Such optical imaging agents would thus enable the diagnostic of cancer or inflammatory diseases, and would be useful for image-guided surgery, for instance during tumour resection.

The present invention thus concerns an optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m, wherein:.

Signal is selected from the group consisting of fluorophores, preferentially fluorophores emitting in the near Infra-red, such as indocyanin green, cyanin-<NUM>, cyanin <NUM>,<NUM>, cyanin-<NUM>, alexa fluors, and derivatives thereof,
a pharmaceutically acceptable salt, solvate or hydrate thereof.

The optical imaging agents of the invention thus exhibit several advantages.

First, the SIGNAL and BIOVECTOR parts of the optical imaging agent of the invention are covalently linked to its SUPPORT part - through the linker L in the case of BIOVECTOR, when present. Such covalent linkage allows for a much-improved selectivity of the optical imaging agent of the invention for inflamed tissues, as compared with agents wherein the BIOVECTOR or the SIGNAL parts are linked to the rest of the imaging agent only through ionic, hydrogen or Van der Waals bonds. Indeed, in the latter case, dissociation of the different parts of the imaging agent could occur in the biological tissues or the body, thus shading doubt as to the accuracy of the observed images. Second, the optical imaging agent of the invention selectively targets markers of inflammation, thus allowing for accurate delimitation of inflamed areas of the body using in vivo or ex vivo optical imaging, in particular fluorescence imaging. Thanks to the portable imaging devices available nowadays, such optical imaging agent can thus be used as a real-time aid to surgery: for instance, after resection of a tumour, it is possible to visualize through optical imaging of the area of interest if all of the tumoral tissues have been removed, or if a further resection is necessary. Other imaging techniques are either more complex to use in this context (in particular MRI, scintigraphy, PET), or less sensitive (echography).

Third, the presence of a SUPPORT linking the SIGNAL part of the optical imaging agent of the invention and its BIOVECTOR part, increases the bioavailability of the optical imaging agent of the invention.

Finally, it is noteworthy that the optical imaging agent of the invention comprises up to <NUM> fluorophores. Indeed, the inventors have surprisingly demonstrated that the presence of more than <NUM> fluorophores have deleterious effect on the intensity of the observed signal, in contrast for instance to what is commonly known for magnetic resonance or scintigraphy imaging agents (see for instance <CIT>).

According to another aspect, the present invention relates to a diagnostic composition comprising at least one optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, and at least one pharmaceutically acceptable excipient as defined in claim <NUM>.

According to another aspect, the present invention relates to the optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m of the invention or the diagnostic composition of the invention for in vivo use as a diagnostic agent, in particular for diagnosing diseases or conditions associated with inflammation.

According to another aspect, the present invention relates to a method of imaging a biological tissue ex vivo, using optical or fluorescence imaging, said biological tissue comprising the optical imaging agent or the diagnostic composition of the invention.

According to another aspect, the present invention relates to a method of ex vivo diagnosing a disease or condition associated with inflammation, comprising ex vivo imaging a biological tissue of a patient in need thereof, in particular a biological tissue obtained through biopsy, said biological tissue comprising the optical imaging agent or the diagnostic composition of the invention.

n and m are chosen so as to avoid any destabilization of SUPPORT. Indeed, the optical imaging agent of the invention should be able to form stable colloidal suspensions in physiologically acceptable solutions.

As used herein, a "stable suspension" is understood as a suspension which does not form any aggregates or does not sediment over a period of at least <NUM> hours, preferably at least <NUM> hours, even more preferably at least <NUM> hours.

As used herein, a "physiologically acceptable solution" is understood as a solution that is nontoxic and suitable for administration to a patient via oral route or parenteral route, in particular by injection, without any undesired side-effects. As such, the osmolarity and osmolality of a physiologically acceptable solution should be controlled so as to avoid any undesired side effects. Examples of solutes suitable for forming a physiologically acceptable solution are water (in particular water for injection, saline, and aqueous solutions such as NaCl <NUM>, PBS, glucose <NUM>%, Hepes <NUM> + glucose <NUM>%, lactose <NUM>%, Hepes <NUM> + lactose <NUM>%).

In the present invention, a "patient" is understood as an animal, preferably a mammal such as a rodent (mice or rats), and preferably a human being.

Numbers n and m are calculated with mass spectrometry experiments. It is known from the skilled person in the art that mass spectrometry characterization is used to assess grafting of fragments on macromolecules.

After the grafting of the SIGNAL and/or the BIOVECTOR fragment, a mass spectrometry analysis provides the additional mass of the grafted molecule compared to the mass of the native SUPPORT. The mass difference allows attribution of a mean grafting value regarding the number of SIGNAL and/or BIOVECTOR fragments present on the molecule.

n is greater than or equal to <NUM> and is less than <NUM>, for instance n is between <NUM> and <NUM>. n is between <NUM> and <NUM>. n can also be greater than or equal to <NUM> and is less than <NUM>, for instance n is between <NUM> and <NUM>, preferably n is equal to <NUM> or <NUM>. According to the present invention the mean grafting value n is greater than or equal to <NUM> and is less than <NUM>. m can be between <NUM> and <NUM>, preferably between <NUM> and <NUM>, more preferably between <NUM> and <NUM>. According to the present invention the mean grafting value m is between <NUM> and <NUM>.

In a particular embodiment, n is between <NUM> and <NUM>, and m is between <NUM> and <NUM>, more preferably between <NUM> and <NUM>.

In general formula (I), SUPPORT represents a physiologically acceptable chemical or biological substrate, that is to say an entity whose administration is compatible with a living being. Typically, SUPPORT is conjugated to SIGNAL and L-BIOVECTOR through all or part of its amino (NH<NUM>) groups located on its surface.

Advantageously, SUPPORT has a particle size of between <NUM> and <NUM>, preferably between <NUM> and <NUM>, more preferably of between <NUM> and <NUM>. In a particular embodiment, SUPPORT has a particle size of between <NUM> and <NUM>.

SUPPORT may itself have biological activity, or on the contrary be totally inert. Advantageously, the SUPPORT is biodegradable.

As used herein, a "biodegradable" entity is an entity which is biodegraded in vivo through the action of proteins such as enzymes, but which however does not degrade spontaneously in a physiological solution in particular at a pH of between <NUM> and <NUM> and at a temperature of between <NUM> and <NUM>, generally for a time period of at most <NUM> days. SUPPORT is preferably selected from proteins. Suitable proteins or protein conjugates are for instance described in <CIT> (see in particular section "<NPL>).

As used herein, "protein" is meant as a macromolecule composed of a string (or sequence) of amino acids linked together by peptide bonds. Proteins suitable for the present invention may have various molecular weights, in particular ranging from <NUM> to <NUM> kDa, preferably from <NUM> to <NUM> kDa. They may be chosen from albumin, ovalbumin, lactalbumin, immunoglobulins, macroglobulins, microglobulins, lipoproteins, circulating hormones and factors, hemocyanins, and derivatives thereof, preferably albumin, in particular a human albumin such as human serum albumin (HSA).

In a preferred embodiment, SUPPORT is a protein, especially albumin, preferably human serum albumin (HSA) or derivatives thereof, with a particle size of between <NUM> and <NUM>, nm preferably of between <NUM> and <NUM>. In particular, use may be made of albumins commercialized by Vialebex or Baxter. Such a small particle size, combined with the fact that optical agent does not aggregate, is of particular interest in terms of biodistribution and bioavailability. According to this embodiment, the optical imaging agent in which SUPPORT is albumin has a size of between <NUM> and <NUM>.

Albumin derivatives are in particular ovalbumin and lactalbumin.

In another embodiment, SUPPORT may also be selected from polysaccharides, nanoparticles (including liposomes), microparticles (including liposomes) or biocompatible polymers.

In the context of the present invention, a polymer is characterized as "biocompatible" if the polymer and its degradation products are non-toxic to the animal or human being to which it is administered, and does not induce adverse effects in the host's body, e.g. immune reaction at the injection site. Biocompatible polymers suitable for the present invention may be chosen from any of the polymers known to those skilled in the art, including poly(N-(<NUM>-hydroxypropyl)methacrylamide) (HMPA), polyethylene glycol (PEG), collagen, polysaccharides, poly(<NUM>-methoxyethylacrylate) (PMEA), polydimethylsiloxane (PDMS), polyvinyl pyrrolidone (PVP), hyaluronic acid (HA), heparan, heparanesulfates, dextrans, dextransulfates, heparins, cyclodextrins and derivatives thereof.

When SUPPORT is a nano- and microparticle, said particle may in particular be made of biodegradable organosoluble polymer.

As used herein, "particles" are understood as particles with an average particle size of between <NUM> and <NUM><NUM>, and in particular between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and in particular between <NUM> and <NUM>. The particles of the invention have a particle size of between <NUM> and <NUM> and typically have an organic core (such as a polymeric core), but may also be liposomes. More specifically, "microparticles" are understood as particles with an average particle size of between <NUM> and <NUM><NUM>, while "nanoparticles" are understood as particles with an average particle size of between <NUM> and <NUM>, in particular between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and in particular between <NUM> and <NUM>, such as between <NUM> and <NUM>.

As biodegradable organosoluble polymer, mention may be made of polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly(ε-caprolactone) (PCL), polyanhydrides, poly (hyaluronan), poly(alkylcyanoacrylates), polyorthoesters, poly (alkylene tartrate), polyphosphazenes, polyamino acids, polyamidoamines, polysaccharides, polycarbonates, polymethylidenemalonate, polysiloxane, polyhydroxybutyrate or poly(malic acid), and copolymers thereof.

Where appropriate, the particle surface may be modified for example to confer to said particle additional properties, such as hydrophilicity, so as to improve their in vivo reactivity, for example by promoting their adsorption of plasma proteins.

Particularly preferred are "targeted nanoparticles", such as targeted liposomes or targeted biocompatible polymers. As used herein, a "targeted nanoparticle" is able to target markers of inflammation, such as Selectin E. In particular, a "targeted nanoparticle" has a biomarker on its surface targeting markers of inflammation. Suitable nanoparticles and microparticles are for instance described in <CIT>.

Conjugates of specific-binding moieties can be used for detecting specific target molecules in biological samples. The specific-binding portion (in the present invention: BIOVECTOR) of such conjugates binds tightly to a target in the sample and the signal-generating portion (herein SIGNAL) is utilized to provide a detectable signal that indicates the presence/and or location of the target.

SIGNAL is a fluorophore, preferably an organic fluorophore, allowing the optical agent of the invention to be detected through optical imaging, in particular fluorescence imaging. In particular, SIGNAL is a hydrophilic fluorophore. Of note, the fluorophore is hydrophilic enough to avoid any precipitation of the optical imaging agent of the invention. SIGNAL is a hydrophilic cyanine derivative, which are well-known fluorophores.

According to the invention, SIGNAL is of formula (Ia) below:
<CHM>
wherein.

Ring A represents a C<NUM>-C<NUM> monocyclic aryl or heteroaryl group or a C<NUM>-C<NUM> fused bicyclic aryl or heteroaryl group, preferably a C<NUM>-C<NUM> monocyclic aryl group or a C<NUM>-C<NUM> fused bicyclic aryl group, such as a phenyl or naphthyl group, and Ring A is optionally substituted by one SO<NUM>-, SO<NUM>Na or SO<NUM>K group, provided that not more than one of Ra1 and Ra2 is substituted by a SO<NUM>- group, provided that the compound of formula (I) comprises not more than one SO<NUM>- group. The compound of formula (I) may have an overall electric charge, namely a positive charge. In such case, it is provided as a salt, in particular a halogenide salt such as a chloride Cl- salt.

The compound of formula (I) is preferably hydrophilic, and as such advantageously contains at least one SO<NUM>- or SO<NUM>Na group, preferably it contains one SO<NUM>- group and SO<NUM>Na group.

In a particular embodiment, SIGNAL is of formula (Ia) below:
<CHM>.

In a particular embodiment, SIGNAL is of formula (Ia1) below:
<CHM>
wherein q, r, Ra1, Ra2, Rb1 and Rb2 are as defined above or below.

In another particular embodiment, SIGNAL is of formula (la2) below:
<CHM>
wherein q, r, Ra1, Ra2, Rb1 and Rb2 are as defined above or below.

In a particular embodiment of formulae (I), (Ia), (la1) and (la2):.

In another particular embodiment, of formulae (I), (Ia), (la1) and (la2):.

In another particular embodiment, SIGNAL is of formula (lb) below:
<CHM>
wherein.

In the compound of formula (I), each ring A is fused with the adjacent pyrrolidine group. Advantageously, SIGNAL is of formula (Ib1) below:
<CHM>
wherein.

In a particular embodiment, SIGNAL is of formula (Ib2) below:
<CHM>
wherein.

In a preferred embodiment, SIGNAL is selected from cyanines and indocyanines of formulae below:
<CHM>
<CHM>
more specifically, SIGNAL is
<CHM>
<CHM>.

In another preferred embodiment, SIGNAL is selected from:
<CHM>
<CHM>
and
<CHM>.

Of note, when excited with light (in particular at a wavelength of between <NUM> and <NUM>, preferably in the "close red domain" or "close infrared domain" i.e. between <NUM> and <NUM>), cyanine emits red light, whereas indocyanine emits green light, through fluorescence phenomena.

The above fluorophores of formula (I) are known in the art and are commercially available, in particular from Luminoprobe.

The BIOVECTOR is a carbohydrate as defined in claim <NUM> targeting selectively a marker of inflammation.

The term "carbohydrate" as used in the present disclosure refers to a monosaccharide or polysaccharide, or nitrogen-derivatives thereof. Monosaccharides are in particular erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, fucose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose. The carbohydrate of the invention is preferably not protected (i.e. it does not contain any O-protecting groups). However, the carbohydrate of the invention may contain one or two sulfate groups, i.e. a OSO<NUM>H or preferably a OSO<NUM>Na group in lieu of a OH group.

Polysaccharides are preferably bi-, tri- or quadrisaccharides, and each monosaccharide group of which it is composed is preferably selected from the monosaccharides listed above.

Nitrogen derivatives of monosaccharides and polysaccharides are understood herein as compounds having a monosaccharide or polysaccharide structure, wherein <NUM> to <NUM> oxygen atoms in a monosaccharide or <NUM> to <NUM> oxygen atoms in a polysaccharide are replaced with a NH group.

Carbohydrates targeting selectively a marker of inflammation are known to the person of skill in the art. In particular, lactose is known not to belong to this category. A typical example is Lewisx pentasaccharide molecule in which the Lewisx trisaccharide is linked to a lactose molecule, however the LewisX-LewisX interaction is due to the Lewisx trisaccharide, but not to lactose.

Preferred carbohydrates targeting selectively a marker of inflammation are mannose, glucose, fucose, sialyl Lewisx (or SLeX), or any mono- or polysaccharide comprised in sialyl Lewisx, such as neuraminic acid or and the trisaccharide of formula:
<CHM>
with Rs being H, SO<NUM>Na or SO<NUM>K, preferably H or
SO<NUM>Na,
and derivatives thereof.

Examples of carbohydrate derivatives are carbohydrates wherein one anomeric OH group is replaced by a O-(C<NUM>-C<NUM>)alkyl-X' group, with X' representing O, NH or S, preferably NH. For instance a carbohydrate derivatives is a carbohydrate wherein an anomeric OH group is replaced by a -O-CH<NUM>-CH<NUM>-NH group. When the carbohydrate comprises a COOH group, a particular carbohydrate derivative is a carbohydrate wherein the group COOH is replaced by a group of formula COO-(C<NUM>-C<NUM>)alkyl-X', such as a COOCH<NUM>CH<NUM>NH group.

In an advantageous embodiment, BIOVECTOR is selected from the group consisting of mannose and sialyl Lewisx (or SLex), and derivatives thereof.

Sialyl Lewisx (or SLex) is indeed an efficient ligand for E-selectin, also known as CD62 antigen-like family member E (CD62E), endothelial-leukocyte adhesion molecule <NUM> (ELAM-<NUM>), or leukocyte-endothelial cell adhesion molecule <NUM> (LECAM2). E-selectin is an adhesion molecule expressed only on endothelial cells activated by cytokines. During inflammation, E-selectin plays an important part in recruiting leukocytes to the site of injury: local release of cytokines IL-<NUM> and TNF-α by damaged cells induces the over-expression of E-selectin on endothelial cells of nearby blood vessels. Leukocytes in the blood expressing the correct ligand will then bind with low affinity to E-selectin. As such, E-selectin is a particularly useful marker of inflammation, in particular overexpressed in the direct vicinity of tumour metastases.

In this context, BIOVECTOR is preferably of formula (II):
<CHM>.

This particular BIOVECTOR may be obtained from the corresponding compound described for instance described in<NPL>). Substructures of Sialyl Lewis X are also useful ligand for E-selectin such as neuraminic acid. In this context, BIOVECTOR is advantageously of formula (III):
<CHM>
wherein R is R<NUM>-X', with R<NUM> and X' as defined above in connection with formula (II).

Also considered is a trisaccharides of formula (IV):
<CHM>
wherein Rs is H or SO<NUM>Na, and R is as defined above in connection with formula (III).

Mannose is also an efficient ligand for macrophages, overexpressed at a site of inflammation, which is useful as a marker of inflammation. A preferred mannose derivative is a compound of formula (V):
<CHM>
wherein R is as defined above in connection with formula (III).

Fucose is also a known to be associated with inflammatory conditions, in particular in cancer. In this context, BIOVECTOR is advantageously of formula (VI):
<CHM>
wherein R is as defined above in connection with formula (III).

Because of the link between diabetes and inflammation in particular, glucose is associated with markers of inflammation. In this context, BIOVECTOR is advantageously of formula (VII), or (VII'):
<CHM>
wherein R is as defined above in connection with formula (III).

In a particular embodiment, L is a linker of formula -C(X)-R<NUM>-Y-, wherein.

Preferably, R<NUM> is a (C<NUM>-C<NUM>)alkyl, preferably a (C<NUM>-C<NUM>)alkyl, optionally interrupted by a
<CHM>
group.

In a preferred embodiment, the optical imaging agent of the invention is of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m, wherein:.

BIOVECTOR is a carbohydrate targeting markers of inflammation selected from the group consisting of mannose, glucose, fucose, neuraminic acid, sialyl Lewisx (SLex), the trisaccharide :.

In this preferred embodiment, SIGNAL is in particular of formula (Ib), (lb1) or (Ib2), wherein, preferably,
p is an integer of between <NUM> and <NUM>, preferably between <NUM> and <NUM>,.

Even more preferably, SIGNAL is indocyanine or sulfo-cyanine <NUM>.

In this preferred embodiment, L is advantageously a linker of formula -C(X)-R<NUM>-Y-, with X being NH, R<NUM> being a (C<NUM>-C<NUM>)alkyl, preferably a (C<NUM>-C<NUM>)alkyl, optionally interrupted by
<CHM>
group, and Y being NH or a -(O)CNH- group.

The present invention also relates to a pharmaceutical or diagnostic composition comprising at least one optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m as defined in the claims, a pharmaceutically acceptable salt, solvate or hydrate thereof, and at least one pharmaceutically acceptable excipient.

The pharmaceutical or diagnostic compositions of the invention are advantageously suitable for administration via oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal route, more preferably via oral route or by injection. The optical imaging agent of the invention can be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals or to humans.

When a solid composition is prepared in the form of tablets, the optical imaging agent of the invention is mixed with a pharmaceutical vehicle and other conventional excipients known to those skilled in the art.

When a composition is formulated for injection, the optical imaging agent of the invention is mixed with a saline solution and other conventional excipients known to those skilled in the art.

The optical imaging agents of the invention can be used in a diagnostic composition at a dose ranging from <NUM>/kg to <NUM>/kg, for instance between <NUM>/kg and <NUM>/kg, in particular between <NUM>/kg and <NUM>/kg, preferably administered in only one dose. However, it may be necessary to use doses outside these ranges, which will then be designed by the person skilled in the art.

In the optical imaging agent of the invention, BIOVECTOR is a carbohydrate targeting selectively a marker of inflammation, preferably mannose, sialyl Lewisx (or SLex) or derivatives thereof, advantageously the derivatives of formula (II) and (III) as defined above.

Therefore, the optical imaging agents of the invention will selectively accumulate in tissues or body areas wherein the targeted markers of inflammation (E-selectin for sialyl Lewisx (or SLex) derivatives, macrophages for mannose derivatives) are overexpressed. The present invention thus also relates to the optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m as defined in the claims or the pharmaceutical or diagnostic composition of the invention for in vivo ise as a diagnostic agent, in particular for diagnosing diseases or conditions associated with inflammation.

The optical imaging agents or diagnostic compositions of the invention are also useful in diagnosing a cardiovascular or inflammatory disease such as myocardial ischemia stroke, inflammatory bowel disease (such as Crohn disease and ulcerative colitis), renal failure, post-operative ileus, brain ischemia, diabetes, diabetic nephropathy, metabolic syndrome, sickle-cell disease, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, neuropathic pain, hypertension, pulmonary arterial hypertension, septicemia, septic or endotoxic shock, hemorrhagic shock, multiple sclerosis, cancer and chronic obstructive pulmonary disease, and arthritic diseases (such as arthritis and osteoarthritis).

Preferably, the optical imaging agent or diagnostic composition of the invention is used for diagnosing cancer (in particular tumours, such as tumours of colorectal cancer), stroke and renal failure.

In a particular embodiment, the disease or condition associated with inflammation is cancer, and in particular tumours, such as tumours of colorectal cancer. In this embodiment, the optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m as defined by the claims or the pharmaceutical or diagnostic composition of the invention is useful as an aid to surgery, in particular as contrast agent in image-guided surgery. In this embodiment, the optical imaging agent or pharmaceutical or diagnostic composition of the invention is used in particular for real time diagnosis, allowing to follow evolution of the resection of tumoral tissues.

In this embodiment, the patient undergoing surgery is a living animal, in particular a human patient, suffering from cancer (such as colorectal cancer), and is in need of surgery for tumour resection. The optical imaging agent or diagnostic composition of the invention is administered to the patient advantageously prior to surgery or during surgery, for instance by injection.

Therefore, the present invention also relates to the optical imaging agent or diagnostic composition of the invention for use in a method of surgically resecting tumours of a patient comprising the following successive steps:.

As used herein, an "area of interest" is understood as an area of tissues or an organ of the patient comprising tumoral tissue to be resected and the vicinity of the tumoral tissue. For instance, in the case of liver cancer, the area of interest may be the whole liver. In the case of colorectal cancer, the area of interest is preferably the colon and/or the rectum, or part of it.

In this particular embodiment, the optical imaging agent or diagnostic composition of the invention thus proves useful in helping resecting the entirety of the tumoral tissues. Such a method using the optical imaging agent or diagnostic composition of the invention as an aid to surgery also allows for resecting the minimum sane tissues, whereas nowadays, in the absence of such method, surgeons tend to resect a wide area of sane tissues around the tumours as a safety measure.

The present disclosure further relates to, but is not part of the present invention, the use of the optical imaging agent of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, for the manufacture of a diagnostic composition, in particular for use for diagnosing diseases or conditions associated with inflammation as detailed above.

In a particular embodiment, the disease or condition associated with inflammation is cancer, and in particular tumours, such as tumours of colorectal cancer. In this embodiment, the disclosure concerns the use of the optical imaging agent of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, for the manufacture of a diagnostic composition for real time diagnosis during surgery, in particular tumour resection.

The present disclosure further concerns a method for diagnosing diseases or conditions associated with inflammation as detailed above, comprising administering an effective amount of the optical imaging agent of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, or the diagnostic composition of the invention, to a patient in need thereof.

In a particular instance, the disease or condition associated with inflammation is cancer, and in particular tumours, such as tumours of colorectal cancer. In this instance, the method of the invention is for real time diagnosis during surgery, in particular tumour resection.

The method of the disclosure (not being part of the present invention) further relates to a method of surgically resecting tumours comprising the following successive steps:.

The present invention concerns the optical imaging agents as described in the claims for image-guided surgery. The principles of image-guided surgery are for instance described in <NPL>. "Indocyanine green fluorescence imaging in the surgical management of liver cancers: current facts and future implications').

The present invention further concerns the optical imaging agents as described in the claims for in vivo use in a method for diagnosing an inflammation, in particular non-invasive optical imaging.

The present invention further concerns a method of imaging a biological tissue ex vivo, using optical or fluorescence imaging, said biological tissue comprising the optical imaging agent or the diagnostic composition of the invention.

The biological tissue is preferably a biopsy sample.

In a first embodiment, the patient is first administered with (an effective amount of) the optical imaging agent of the invention or the diagnostic composition of the invention, and the biopsy is then carried out to take a sample of tissue, and the tissue of the biopsy is then fixed under conditions selected by the one of skill in the art so as to interfere neither with the SIGNAL part of the optical imaging agent of the invention, nor in the BIOVECTOR-Marker of inflammation recognition. In other words, the fixation step of the tissue does not degrade it, so that imaging of the tissue faithfully reflects the state of the remaining tissue in the body.

In this first embodiment, the biopsy tissue is then imaged using an optical imaging device (more specifically a fluorescence imaging device). Devices allowing for 2D or 3D imaging may be used, depending on the thickness of the biopsy, and the condition or disease associated with inflammation from which the patient is suffering.

Examples of 3D optical imaging techniques are for example optical tomography.

In a second embodiment, the patient is not administered with the optical imaging agent of the invention or the diagnostic composition of the invention prior to the biopsy. In this second embodiment, the biopsy tissue is thus subjected to the optical imaging agent of the invention or the diagnostic composition of the invention for a time sufficient to allow the optical imaging agent to bind to the targeted marker of inflammation (e.g. E-selectin in the case of BIOVECTOR of formula (II) as defined above, or macrophages in the case of BIOVECTOR of formula (III) as defined above), and then rinsed. Then, the tissue of the biopsy is then fixed under conditions selected by the one of skill in the art so as to interfere neither with the SIGNAL part of the optical imaging agent of the invention, nor in the BIOVECTOR-Marker of inflammation recognition. In other words, the fixation step of the tissue does not degrade it, so that imaging of the tissue faithfully reflects the state of the remaining tissue in the body.

In this second embodiment, the biopsy tissue is then imaged using an optical imaging device (more specifically a fluorescence imaging device). Devices allowing for 2D or 3D imaging may be used, depending on the thickness of the biopsy, and the condition or disease associated with inflammation from which the patient is suffering.

The present invention further relates to a method of diagnosing ex vivo a disease or condition associated with inflammation as detailed above, comprising ex vivo imaging a biological tissue of a patient in need thereof, in particular a biological tissue obtained through biopsy as explained above, said biological tissue comprising the optical imaging agent or the diagnostic composition of the invention.

The present disclosure further provides a process for preparing the optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m as defined above, a pharmaceutically acceptable salt, solvate or hydrate thereof. the process of the invention preferably comprises the following successive steps:.

Optionally, the process comprises a step d) of washing the optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m , preferably with water or a NaCl aqueous solution.

The succinimide derivative of SIGNAL are easily accessible from the corresponding SIGNAL compound using derivatization methods well known in the art.

In the same way, compounds of formula H-L-BIOVECTOR are easily accessible to the one of skill in the art from the corresponding BIOVECTOR, using methods well known in the field of carbohydrate chemistry, especially when L is of formula-C(X)-R<NUM>-Y-, with X being O, R<NUM> being a (C<NUM>-C<NUM>)alkyl, optionally interrupted by <NUM> to <NUM> groups selected from a heteroatom such as O or NH, a -C(O)- group, a -NHC(O)- group, a -(O)CNH- group, and a
<CHM>
group , and Y a NH or a -(O)CNH- group.

BIOVECTOR is easily accessible from the corresponding carbohydrate, in particular when BIOVECTOR is of formula (II), (III), (V), (VI) and (VII), which may be easily prepared from Sialyl Lewisx, neuraminic acid, mannose, fucose and glucose, respectively.

The compounds of formula (IV) may be easily prepared from the corresponding trisaccharide, which may be prepared as follows:.

The thus obtained protected azido-trisaccharide may be deprotected as follows:
<CHM>.

Monosaccharide
<CHM>
is obtained for instance as follows:
<CHM>
<CHM>
is for instance obtained as described in <NPL>.

The compounds BIOVECTORS-LH may then for instance be prepared as follows:
<CHM>
functionalization of the protein using this compound preferably occurs through a Michael addition of alpha,beta-unsaturated double bond of the maleimide moiety by a free SH group present on the surface of the protein (preferably the albumin). The free SH group is introduced on the albumin for instance by reacting albumin with the Traut's reagent (<NUM>-iminothiolane).

The present invention encompasses only stable compounds. In this regard, when "isomers" are referred to, only stable isomers are considered.

Within the groups, radicals or fragments defined in the description and the claims, the number of carbon atoms is specified inside the brackets. For example, (C<NUM>-C<NUM>)alkyl designates an alkyl group or radical having <NUM> to <NUM> carbon atoms.

In the formulas, <IMG> indicates the bond linked to the rest of the molecule.

As used herein, a "-(C<NUM>-C<NUM>)alkyl" designates an acyclic, saturated, linear or branched hydrocarbon chain comprising <NUM> to <NUM> carbon atoms. Examples of -(C<NUM>-C<NUM>)alkyl groups include methyl, ethyl, propyl, butyl, pentyl or hexyl. Unless explicitly stated, the definitions propyl, butyl, pentyl and hexyl include all possible isomers, in particular structural isomers. For example, butyl comprises n-butyl, iso-butyl, sec-butyl and tert-butyl.

The term "aryl" designates an aromatic, monocyclic ring that may be fused with a second saturated, unsaturated or aromatic ring. The term aryl include, without restriction to the following examples, phenyl, indanyl, indenyl, tetrahydronaphtyl and dihydronaphtyl. The most preferred monocyclic aryl is phenyl, while the most preferred bicyclic fused aryl is naphthyl. The aryl group may be substituted, preferably with one or more groups independently selected from the group consisting of alkyl, alkoxy, halogen, hydroxyl, amino, nitro, cyano, trifluoro, carboxylic acid or carboxylic ester.

The term "heteroaryl" designates aromatic, monocyclic ring that may be fused with a second saturated, unsaturated or aromatic ring where one or more carbon atoms have been replaced with one or more heteroatoms chosen from among N, O and S. Unless explicitly stated, the term "heteroaryl" includes all possible isomers, in particular position isomers. Examples of monocyclic heteroaryl groups include furyl, thienyl, imidazolyl, pyridyl, pyrrolyl, N-alkyl pyrrolyl, pyrimidinyl, pyrazinyl, tetrazolyl, triazolyl and triazinyl. Examples of fused bicyclic heteraryls include indolyl, indolinyl, benzofuryl, benthienyl, quinoleine, isoquinoleine. The heteroaryl group may be substituted, preferably with one or more groups independently selected from the group consisting of alkyl, alkoxy, halogen, hydroxyl, amino, nitro, cyano, trifluoro, carboxylic acid or carboxylic ester. Preferred heteroaryls are those having <NUM> or <NUM> atoms in the ring, such as indolyl, pyrrolyl, pyridinyl, pyrrazolyl, triazolyl, furanyl or thienyl.

For the purpose of the invention, the term "pharmaceutically acceptable" is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and non-toxic, for a pharmaceutical use.

The term « pharmaceutically acceptable salt, hydrate of solvate » is intended to mean, in the framework of the present invention, a salt of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound. Such salts comprise:.

In the present invention, the term "size" is understood as the hydrodynamic diameter of the particles.

In the present description, the carbohydrate Sialyl Lewisx is abbreviated without any distinction to Slex or SLX.

The following examples are given for illustrative purpose only.

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

Albumin (<NUM>) is coupled to N-N-disulfonate-cyanin-<NUM>-N-hydroxysuccinimide :
<CHM>
available from luminoprobe (<NUM> equivalents per amine) in PBS and left for <NUM> minutes under gentle stirring. The mixture is washed with an ultrafiltration device with a 50kDa cut off at <NUM> rpm during <NUM> minutes at <NUM>. Sialyl lewis-X-maleimide is then coupled to the labelled albumin in PBS/EDTA at room temperature during <NUM> minutes in presence of <NUM>-iminothiolane hydrochloride (<NUM> equivalent per amine). A washing step is performed to remove the excess of iminothiolane and obtain the labelled targeting albumin HSA-Slx in NaCl <NUM>,<NUM>%. The full functionalisation of albumin was confirmed by mass spectrometry with a mass of <NUM> for the imaging agent (<FIG>).

Sialyl lewis-X-maleimide is obtained as follows. To Sialyl Lewis X (described in <NPL>) is added of N-Succinimidyl <NUM>-maleimidopropionate (hereafter NHS-maleimide):
<CHM>
and triethylamine at room temperature, the reaction mixture is stirred for <NUM> hours at room temperature. The reaction mixture is then evaporated and the crude product is purified using column chromatography on silica gel.

Albumin (<NUM>) is coupled to N-N-disulfonate-cyanin-<NUM>-N-hydroxysuccinimide (<NUM>,<NUM>) in <NUM> PBS and left for <NUM> minutes under gentle stirring. The mixture is washed with an ultrafiltration device with a 50kDa cut off at <NUM> rpm during <NUM> minutes at <NUM>. 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.

Mannose-maleimide is then coupled to the labelled albumin HSA - CY5 in PBS/EDTA at room temperature during <NUM> minutes in presence of <NUM>-iminothiolane hydrochloride (<NUM> equivalent per amine). A washing step is performed to remove the excess of iminothiolane and obtain the labelled targeting albumin in NaCl <NUM>,<NUM>%. The expected mass of the imaging agent was confirmed by mass spectrometry (M <NUM>) (<FIG>). The reaction of <NUM> equivalents of cyanin gave the highest intensity of fluorescence as referred to <NUM> equivalents (<FIG>).

Mannose-maleimide
<CHM>
is obtained as follows. <NUM>-aminoethylmannopyranoside
<CHM>
(<NUM>, <NUM> mmol) is dissolved in DMF.

NHS-maleimide (<NUM>, <NUM> 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 : <NUM>/<NUM>). <NUM> of purified Mannose-maleimide is obtained.

The in vitro experiments were performed with bEnd. <NUM> mus musculus brain endothelial cells (CRL-<NUM>™) and CT26. WT mus musculus colon carcinoma cells (CRL-<NUM>™) provided by ATCC®. Those cells were cultured in DMEM-Dulbecco's Modified Eagle Medium (<NUM>-<NUM>, Gibco Thermo Fisher) added by <NUM>% of fetal bovine serum (<NUM>, Gibco Thermo Fisher) and <NUM>% of Penicillin-Streptomycin (<NUM>,<NUM> U/mL, <NUM>, Gibco Thermo Fisher) antibiotics. A suspension of Lipopolysaccharide stimulated cells (<NUM>,<NUM>/mL, <NUM>, <NUM>), were collected and prepared in binding buffer (<NUM> Tris HCI, <NUM> NaCl, <NUM> CaCl2, <NUM> MgCl2, <NUM> MnCl2,<NUM>% BSA in water) at <NUM><NUM> cell per milliliter. An incubation of <NUM> at <NUM> was realized in order to saturate unspecific binding. For evaluation of E-selectin expression (<FIG>) cells were incubated successively with purified rat anti-Mouse CD62E antibody (1µg/mL, <NUM> BD Pharmingen™) in PBS/BSA <NUM>% and with goat anti-rat IgG-Alexa Fluor ®<NUM> (1µg/ml, ab150157 Abcam) for <NUM> à <NUM>. To study cellular binding and internalization, cells were incubated with 30ng of HSA or HSA-SLX for <NUM> at <NUM> (<FIG>) or <NUM> (<FIG>).

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

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.

BalbC female mice (from janvier labs) were implanted with <NUM><NUM> of CT26 tumour fragment in subcutaneous way. Fifteen day after implantation, mice were anesthetized with a mixture of ketamine (<NUM>/kg, Clorketam® <NUM><NUM> Vetoquinol) and xylazine (<NUM>/kg, ROMPUN® <NUM> % Bayer), 200µl of HSA-SLX-Cy5 (<NUM> /ml) suspension was then injected into the tail vein. Twenty four hour after injection, an evaluation of image guided surgery (<FIG>) was realized under mice anaesthesia with Fluobeam® open Imaging system for in vivo near infrared fluorescence imaging (λ ex <NUM>, λ em > <NUM>)λ.

Female balbc mice (from janvier labs) were anesthetized with ketamine / xylazine mixture, <NUM> mice per condition were injected with HSA-CY5 and HSA-Slx suspension (<NUM>/ml) in the tail vein of Balbc mice <NUM> days after CT26 tumour implantation. The biodistribution kinetic of formulation was recorded by Fluobeam® system as function of time (<FIG>). 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>) were imaged and quantified by PhotonIMAGER™ 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.

Collagen Arthritis model was induced in DBA/<NUM> 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 (<NUM>/ml) in the tail vein. In vivo kinetic of the formulation was recorded by Fluobeam® system at different time points (<FIG>). Twenty for hours after probe injection colocalization signal between probes and arthritis articulation could be evaluated by PhotonIMAGER™ optima (Biospace lab) after luminol injection (<FIG>).

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 <NUM>, but substituting lactose for mannose) or HSA-Mannose (HSA-M-CY5) suspension (<NUM>/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>).

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.

Separation and analysis were performed on a LC-<NUM> 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 <NUM> column provided by Showa Denko (Japan). Between the auto-sampler and HPLC pump, mobile phase was filtered in-line by a <NUM> durapore PVDF membrane in a PEEK in-line filter. Detection was realized using UV absorbance at a wavelength of <NUM>. Concomitantly, refractive index quantifications were performed using white light. Mobile phase was CH3COONH4 <NUM> NaN3 <NUM>% 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 <NUM> wavelength laser. Data analysis was achieved on Astra <NUM>, Wyatt Technology.

HSA-SLX corresponds to the optical imaging agent described in example <NUM>.

HSA-SLX2 corresponds to the optical imaging agent described in example <NUM>, 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:
<CHM>
wherein Rs is SO<NUM>Na.

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.

Female BALB/cJRj <NUM> weeks old mice (Janvier labs, Le Genest-Saint-Isle, France) were anesthetized with a mixture of <NUM> kg-<NUM> of ketamine (IMALGENE <NUM> Boehringer Ingelheim, France) and <NUM> kg-<NUM> of xylazine (Rompun™, Bayer, France) and injected by an intravenous route with <NUM>µl of HSA-SLX or HSA-SLX2 solutions, with both concentrations at <NUM> mL-<NUM>. 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 (<NUM>, λex <NUM>, λem <NUM>).

The images were processed using the M3vision software. The result is expressed as percent of the fluorescence found per mice (<FIG>).

HSA SLX and HSA SLX <NUM> are as described in Example <NUM>.

Claim 1:
Optical imaging agent of formula (SIGNAL)n-SUPPORT-(L-BIOVECTOR)m, wherein:
SUPPORT represents a physiologically acceptable chemical or biological substrate, with a particle size of between <NUM> and <NUM>,
SIGNAL is a fluorophore of formula (I):
<CHM>
wherein
q is <NUM> or <NUM>,
r is <NUM> or <NUM>,
Rb1 and Rb2 are H or a C<NUM>-C<NUM> group, or are bridged to form a -CH<NUM>-CH<NUM>-CH<NUM>- alkylene group (only when q and r represent <NUM>),
Ra1 and Ra2 are identical or different and are independently a (C<NUM>-C<NUM>)alkyl group, optionally substituted by a SO<NUM>- group, a SO<NUM>K group, a SO<NUM>Na group, or a COOH group, provided that not more than one of Ra1 and Ra2 is substituted by a SO<NUM>- group.
Ring A represents a C<NUM>-C<NUM> monocyclic aryl or heteroaryl group or a C<NUM>-C<NUM> fused bicyclic aryl or heteroaryl group, preferably a C<NUM>-C<NUM> monocyclic aryl group or a C<NUM>-C<NUM> fused bicyclic aryl group, such as a phenyl or naphthyl group, and Ring A is optionally substituted by one SO<NUM>- or SO<NUM>Na group, provided that not more than one of Ra1 and Ra2 is substituted by a SO<NUM>- group,
provided that the compound of formula (I) comprises at least one SO<NUM>- or SO<NUM>Na group, but not more than one SO<NUM>- 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 CI- salt,
L is a linker of formula -C(X)-R<NUM>-Y-, with
X being O, NH or S,
R<NUM> being a (C<NUM>-C<NUM>)alkyl group, preferably a (C<NUM>-C<NUM>)alkyl group, optionally interrupted by <NUM> to <NUM> groups selected from -O-, -NH-, -C(O)-, -NHC(O)-, -(O)CNH-, -C(O)-NH-N=C-, -N=C- , and
<CHM>
Y being NH or -(O)CNH-, and
BIOVECTOR is a carbohydrate able to target markers of inflammation of formula (II):
<CHM>
wherein R<NUM> represents a (C<NUM>-C<NUM>)alkyl group, preferably a (C<NUM>-C<NUM>)alkyl group,
and X' represents a heteroatom such as O, S or NH, preferably NH, or
a compound of formula (III) or (IV) or (V) or (VI) or (VII):
<CHM>
<CHM>
wherein R is R<NUM>-X', with R<NUM> and X' as defined above in connection with formula (II),
and Rs is H, SO<NUM>K or SO<NUM>Na,,
wherein the mean grafting value n is greater than or equal to <NUM> and is less than <NUM>,
wherein the mean grafting value m is between <NUM> and <NUM>, a pharmaceutically acceptable salt, solvate or hydrate thereof.