Methods for labeling eukaryotic cells from multicellular organisms and for treating and / or diagnosing cancer using modified monosaccharide compounds

Abstract

The present invention relates to modified monosaccharide compounds implemented in methods for labeling and / or detecting eukaryotic cells from multicellular organisms. The present invention also relates to such modified monosaccharide compounds implemented in methods for identifying or isolating cancer cells, diagnosing cancer or for use in cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of medicine, particularly oncology. It relates to modified monosaccharide compounds for use in methods for labeling and / or detecting and / or targeting eukaryotic cells from multicellular organisms. The invention also relates to such modified monosaccharide compounds for use in methods for identifying or isolating cancer cells, diagnosing cancer, or in cell therapy. Background Art

[0002] Carbohydrates are important as signaling molecules and for cellular recognition events. Indeed, they can generate multivalent interactions with carbohydrate recognition proteins (CRPs) and serve as probes in living organisms. Therefore, carbohydrates offer numerous opportunities in disease diagnosis and treatment. Consequently, the development of carbohydrate-based bioactive compounds and sensors has become an active area of research. Click chemistry is an efficient and modular synthetic approach for preparing functional carbohydrate derivatives. A review by He et al. reports on several therapeutic and diagnostic carbohydrate derivatives prepared by CuAAC (Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition) click chemistry (Carbohydrate Research 429 (2016) 1-22). Copper-free click chemistry using strain-promoted azide-alkyne cycloaddition has also been described for cancer cells. Specific sugars containing clickable groups (e.g., triacetyl N-azidoacetylmanosamine (Ac3ManNAz) analogs) are metabolized by cancer cells due to the cleavage of the sugar analogs by specific overexpressed enzymes. The sugar is thereby specifically metabolized and incorporated into the cancer cell membranes.

[0003] Even though various carbohydrate / monosaccharide click chemistries have been described for therapeutic and diagnostic applications, these click chemistry methods and the compounds used therein appear to be cytotoxic and / or non-selective for specific cells, such as cancer cells. The cytotoxicity of these compounds necessitates the use of low, non-toxic concentrations, which makes them less effective.

[0004] WO2013 / 1077559 describes modified monosaccharide compounds, such as the specific compound 8-azido-3,8-dideoxy-D-manno-octulose acid (also referred to herein as "KDO-N3"), for use in methods for labeling specific living microorganisms. The labeled living microorganisms are limited to unicellular prokaryotic microorganisms (bacteria).

[0005] WO2016 / 177712 also describes modified monosaccharide compounds, such as the specific compound 5-azido-5-deoxy-D-arabinofuranosyl (also referred to herein as "Ara-N3"), for use in methods for labeling specific living microorganisms. The labeled living microorganisms are limited to unicellular prokaryotic microorganisms (bacteria) and unicellular eukaryotic microorganisms (yeast, fungi, and amoeba).

[0006] However, it has not been explained so far how and where the monosaccharide compounds are assimilated by the cell membrane of the microorganisms.

[0007] There is a constant need to find and develop new candidates, particularly for labeling and detecting eukaryotic cells from multicellular organisms, to provide methods for identifying or isolating cancer cells, and to provide methods for diagnosis or cell therapy, particularly in the field of cancer. Summary of the Invention

[0008] The present invention is based on a monosaccharide compound of formula (I) or a precursor thereof:

[0009]

[0010] wherein X is a reactive group that allows for the covalent attachment of another compound, such as a label or an anticancer drug or a particle containing an anticancer drug, via a click chemistry reaction. The resulting conjugate can therefore be used in methods for labeling or detecting eukaryotic cells from multicellular organisms, methods for identifying or isolating cancer cells, or methods for treating cancer.

[0011] More specifically, the present inventors have discovered that monosaccharide compounds of formula (I), particularly Ara-N3, undergo assimilation with eukaryotic cells from multicellular organisms. They have also discovered that this assimilation in tumor eukaryotic cells is different from that in non-tumor eukaryotic cells and is more significant than that in non-tumor cells (particularly bladder cancer, blood cancer, skin cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, lung cancer, muscle cancer, lymphocyte cancer, prostate cancer, stomach cancer, and breast cancer compared to non-cancerous cells). Therefore, monosaccharide compounds of formula (I) and their precursors can also be used as cancer probes and markers, which can be used to identify, isolate or target cancer cells, and / or for diagnosing cancer in a subject.

[0012] Thus, one aspect of the present invention is a method for labeling or detecting or targeting eukaryotic cells from a multicellular organism, preferably an in vitro method, comprising the steps of:

[0013] a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound or a precursor thereof;

[0014] b) contacting the sample of step (a) with a compound bearing a first reactive group, optionally in the presence of copper; and

[0015] c) optionally detecting the binding of the compound of step (b) to the monosaccharide of step (a) to detect eukaryotic cells;

[0016] wherein the at least one modified monosaccharide compound has the following formula (I):

[0017]

[0018] wherein X is a second reactive group, the first reactive group and the second reactive group are capable of reacting together in a click chemistry reaction to obtain the compound of step (b) bonded to the monosaccharide. In a preferred embodiment, the first reactive group is an alkynyl group and the second reactive group X is an azido group (-N3).

[0019] Another aspect of the invention is a kit for carrying out the method of labelling, detecting or targeting a eukaryotic cell as defined herein, comprising:

[0020] - a modified monosaccharide compound of formula (I) or a precursor thereof, preferably formula (I'), and

[0021] - a compound bearing a first reactive group.

[0022] Another aspect of the invention is a method for identifying or isolating cancer cells or diagnosing cancer in a subject, comprising performing a method for labelling, detecting or targeting eukaryotic cells from a multicellular organism as defined herein from said subject, preferably in a biological sample from said subject.

[0023] Another aspect of the present invention is a composition comprising a eukaryotic cell having at least one modified monosaccharide compound of formula (I) or a precursor thereof (preferably formula (I')) present on its surface, as defined herein, the monosaccharide compound or precursor thereof being operably linked or not to an anticancer drug or a particle comprising at least one anticancer drug. Another aspect is a pharmaceutical composition comprising such a cell. Another aspect is such a pharmaceutical composition for use in treating cancer, particularly by cell-based therapy. Another aspect is such a pharmaceutical composition for use in diagnosing cancer.

[0024] Another aspect of the invention is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition as defined herein, comprising a eukaryotic cell having on its surface at least one modified monosaccharide compound of formula (I) or a precursor thereof (preferably formula (I')), as defined herein, operably linked or not to an anticancer drug or a particle comprising at least one anticancer drug.

[0025] Another aspect of the invention is the use of a modified monosaccharide compound as defined herein, or a precursor thereof, optionally together with a compound carrying a first reactive group, for use in medical imaging or diagnosis, preferably for use in the diagnosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Ex vivo fluorescence signal ratio (tumor / muscle ratio) of tumor and skeletal muscle obtained from Panc-1 tumor-bearing mice with or without intravenous injection of Ara-N3 and fluorophore. Detailed Description of the Invention

[0028] definition

[0029] According to the present invention, the following terms have the following meanings:

[0030] "Multicellular organisms" include any organism comprising more than one cell. Multicellular organisms are derived from or are, for example, plants or animals, preferably mammals, more preferably humans.

[0031] As used herein, the terms "patient" and "subject" are used interchangeably and include humans and animals, more particularly humans.

[0032] "Eukaryotic cells from multicellular organisms" are cells that have a nucleus within a membrane, unlike prokaryotes, and are derived from multicellular organisms, such as animal or plant cells. Animal and plant cells are the most familiar eukaryotic cells from multicellular organisms. In the context of the present invention, "eukaryotic cells" include cancer cells and normal cells (or non-tumor cells and tumor cells).

[0033] Cancer cells are cells that divide indefinitely, forming solid tumors or filling the blood with abnormal cells. Thus, it can be a solid cancer or a hematopoietic cancer, such as lymphoma or leukemia.

[0034] As used herein, the term "cancer" or "tumor" refers to the presence of cells that have typical characteristics of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain typical morphological characteristics. The term refers to any type of malignant tumor (primary or metastatic). Typical cancers are solid cancers or hematopoietic cancers, such as breast cancer, brain cancer, stomach cancer, liver cancer, skin cancer, prostate cancer, pancreatic cancer, esophageal cancer, sarcoma, ovarian cancer, endometrial cancer, bladder cancer, cervical cancer, rectal cancer, colon cancer, kidney cancer, lung cancer or ORL cancer, pediatric tumors (neuroblastoma, glioblastoma multiforme), lymphoma, carcinoma, glioblastoma, hepatoblastoma, leukemia, myeloma, seminoma, Hodgkin's tumor, or hematologic malignancies.

[0035] The present invention relates to a method for labeling or detecting or targeting eukaryotic cells from a multicellular organism, the method comprising the following steps:

[0036] a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound or a precursor thereof;

[0037] b) contacting the sample of step (a) with a compound bearing a first reactive group, optionally in the presence of copper; and

[0038] c) optionally comprising detecting the bonding of the compound of step (b) to the monosaccharide of step (a);

[0039] wherein the at least one modified monosaccharide compound has the following formula (I):

[0040]

[0041] Wherein X is a second reactive group, and the first and second reactive groups can react together in a click chemistry reaction. The reaction between the first and second reactive groups allows the compound of step (b) to be bonded to the monosaccharide of formula (I).

[0042] Click chemistry is a well-known method for attaching probes or substrates of interest to specific biomolecules, such as the modified monosaccharide compounds according to the present invention. Azide-alkyne cycloaddition is a well-known so-called click chemistry reaction, in which an azide group reacts with an alkyne group in the presence or absence of a copper catalyst to form a triazole. The alkyne group may or may not be strained.

[0043] This azide alkyne cycloaddition reaction can be carried out under copper-catalyzed conditions in the presence of a ligand, preferably a tris-triazole ligand such as TGTA (tris((1-(-D-pyranosylglucopyranosyl)-1[1,2,3]-triazol-4-yl)methyl)amine) or TBTA (tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine). Other suitable ligands that are frequently used are tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanesulfonic acid (BTTES), tris((1-(((ethyl)carboxymethyl)-(1,2,3-triazol-4-yl))methyl)amine, bathophenanthroline disulfonate or tris(2-benzimidazolylmethyl)amine.

[0044] Alternatively, the azide alkyne cycloaddition can be performed in the absence of copper if strained alkynes such as azadibenzocyclooctyne (ADIBO, DIBAC, or DBCO) or tetramethoxydibenzocyclooctyne (TMDIBO) are used. Other suitable strained alkynes commonly used in copper-free reactions include cyclooctyne (OCT), arylcyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiophene (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxadibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne.

[0045] Other reactive groups and other reactions can also be used in click chemistry, such as: Staudinger Ligation (first reactive group = azide, second reactive group = phosphine), copper-free click chemistry (first reactive group = azide, second reactive group = constrained alkyne (intracyclic alkyne)), carbonyl condensation (first reactive group = aldehyde or ketone, second reactive group = hydrazide or amine), thiol-ene click chemistry (first reactive group = thiol and second reactive group = olefin), nitrile-oxide-ene click chemistry (first reactive group = nitrile oxide or aldehyde, oxime or hydroxymoylchloride or chlororoxime, second reactive group = olefin or alkyne), nitrile imine-ene click chemistry (first reactive group = nitrile imine or aldehyde, hydrazone or hydrazonoyl chloride ...azone chloride) or chlorohydrazone, second reactive group = alkene or alkyne), reverse electron demand Diels-Alder ligation (first reactive group = alkene, second reactive group = tetrazine), isonitrile-tetrazine click chemistry (first reactive group = isonitrile, second reactive group = tetrazine), Suzuki-Miyaura coupling (first reactive group = aryl halide, second reactive group = aryl boronate), His-tag (first reactive group = oligohistidine, second reactive group = nickel complex or nickel ligand).

[0046] Monosaccharide compounds and their precursors

[0047] According to the present invention, the modified monosaccharide compound of formula (I) includes a reactive group X (second reactive group) suitable for reacting in a click chemistry reaction, preferably in an azide alkyne cycloaddition reaction. Therefore, X includes any reactive group capable of reacting with another reactive group by a click chemistry reaction, such as a reactive group as defined above. In a specific embodiment, X includes any group consisting of or carrying an azide (-N3) and a group consisting of or carrying a strained or unstrained alkynyl (-C≡C-). In a preferred embodiment, X is an azido (-N3).

[0048] In a specific embodiment of the present invention, the modified monosaccharide compound of formula (I) includes its diastereomers. In another specific embodiment, the modified monosaccharide compound of formula (I) is selected from the group consisting of:

[0049]

[0050] In a preferred embodiment, the at least one modified monosaccharide compound of formula (I) is 5-azido-5-deoxy-D-arabinofuranosyl (Ara-N3), in particular having the following formula:

[0051]

[0052] In a further embodiment of the present invention, at least one modified monosaccharide compound of formula (I) is a precursor of the modified monosaccharide compound. More specifically, the precursor has formula (I'):

[0053]

[0054] Wherein X is a second reactive group as defined above, preferably an azide group (-N3), said first and second reactive groups can react together in a click chemistry reaction.

[0055] In a further embodiment, the precursor of formula (I') includes its diastereomers. In a further embodiment, the precursor of formula (I') is selected from the group consisting of:

[0056]

[0057] In a preferred embodiment, the precursor of formula (I') is 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose having the formula:

[0058]

[0059] The modified monosaccharide compound of formula (I) or its precursor can be used in any concentration according to the present invention because it is not toxic to cells. According to a specific embodiment, the concentration of the monosaccharide compound of formula (I) of the present invention or its precursor can vary between 10 μM and 100 mM, preferably 1 mM to 50 mM, more preferably 1 mM to 20 mM.

[0060] Compounds with a first reactive group

[0061] The compound with the first reactive group includes or is a directly detectable moiety, or includes or is an indirectly detectable moiety. Without being bound by any theory, the cell is coupled to the compound with the first reactive group due to a click reaction with the second reactive group of the monosaccharide of step (a) that has been assimilated to the cell membrane in step (a). A detectable moiety (or label) is a moiety that can be detected by techniques known to those skilled in the art, such as fluorescence, colorimetry, or luminescence. Thus, the imaging technique can be fluorescence, magnetic resonance imaging, or computed tomography.

[0062] According to one embodiment, the compound may comprise or may be a detectable moiety, i.e. a moiety consisting of or carrying a detectable substance (or label), i.e. a substance that can be detected by techniques known to those skilled in the art, such as fluorescence, colorimetry or luminescence.

[0063] According to another embodiment, the compound carrying a first reactive group comprises or is an indirectly detectable portion, which is a first ligand (or more specifically, a first binding protein carrying the first reactive group) and the detection and / or immobilization in step (c) as described in detail below can be carried out by contacting the eukaryotic cell coupled to the first ligand (or more specifically, the first binding protein) with a second ligand (or second binding protein) that specifically reacts or binds to the first ligand (or more specifically, the first binding protein).

[0064] More specifically, the compound is a first ligand carrying the first reactive group, preferably biotin, and in step c), the eukaryotic cells coupled to the first ligand are detected by reaction of the eukaryotic cells with an antibody or another protein specific for the first ligand, wherein the antibody carries a detectable substance or moiety, preferably a fluorescent dye or a luminescent molecule or an enzyme.

[0065] The detectable substance or moiety can be selected from dyes, radioactive labels and affinity tags. In particular, the dye can be selected from fluorescent, luminescent or phosphorescent dyes, preferably dansyl, fluorescein, acridine, rhodamine, coumarin, BODIPY and cyanine dyes. More specifically, the fluorescent dye can be selected from dyes sold by Thermo Fisher, such as Alexa Fluor dyes, Pacific dyes or Texas Red or dyes of cyanine 3, 5 and 7 provided by other suppliers. In particular, the dye with azide for CuAAC is commercially available Alexa Fluor. 488, 55, 594 and 647 and TAMRA (tetramethylrhodamine). In a second aspect, the detectable substance or moiety (or label) can be an affinity tag. Such an affinity tag can, for example, be selected from biotin, His-tag, Flag-tag, strep-tag, sugar, lipid, sterol, PEG-linker and cofactor. In a specific embodiment, the detectable substance is a biotinylated label. Biotin connected to an azide is commercially available (biotin azide). In a preferred embodiment, the label is a fluorescent label.

[0066] According to the present invention, the compound with a first reactive group comprises a first reactive group that is complementary to a second reactive group X as defined above for reacting together in a click chemistry reaction, preferably an azide-alkyne cycloaddition reaction. In a specific embodiment, the first reactive group of the compound comprises any group consisting of or carrying an azide (-N3) group and a group consisting of or carrying a strained or unstrained alkynyl (-C≡C-) group.

[0067] In the above list of groups involved in the reaction, the first reactive group and the second reactive group X can be replaced. All of the above chemical reactions will produce a covalent bond. For example, when X is an azido group (-N3) or a group with an azido group, the first reactive group is an alkyne group or a group with an alkynyl group. When X is an alkyne group or a group with an alkynyl group, the first reactive group is an azido group (-N3) or a group with an azido group. In a preferred embodiment of the present invention, the second reactive group X is an azido group (-N3) and the first reactive group is an alkynyl group (-C≡C-).

[0068] Labeling, detection, or targeting methods

[0069] Step a) of the method for labeling, detecting or targeting eukaryotic cells from multicellular organisms comprises contacting a sample containing eukaryotic cells with at least one modified monosaccharide compound of formula (I) or a precursor thereof containing a reactive or functional group X. Such a contacting step a) allows the incorporation of at least one modified monosaccharide compound of formula (I) or a precursor thereof into the membranes of the eukaryotic cells from multicellular organisms, more particularly onto the surface of the cells. Such a process may correspond to the assimilation of at least one modified monosaccharide compound of formula (I) by the eukaryotic cells. Thus, the cells present at least one modified monosaccharide compound of formula (I) on their surface or membrane.

[0070] Step b) comprises contacting the sample of step (a) (wherein at least one modified monosaccharide compound of formula (I) or a precursor thereof is incorporated into the membrane of a eukaryotic cell) with a compound comprising a first reactive group as defined above. Such step b) allows a click chemistry reaction to occur between the first reactive group of the compound and the second reactive group X of at least one modified monosaccharide compound of formula (I) or a precursor thereof (preferably formula (I')), thereby providing a coupled eukaryotic cell from a multicellular organism that is labeled or targeted, or can subsequently be labeled or targeted (as described in detail above).

[0071] A preferred embodiment of the present invention is a method for labeling, detecting or targeting eukaryotic cells from a multicellular organism, the method comprising the steps of:

[0072] a) contacting a sample comprising eukaryotic cells with Ara-N3; and

[0073] b) contacting the sample with a compound comprising a first reactive group, optionally in the presence of copper.

[0074] Those skilled in the art know how to implement step (a) or (b). According to a specific embodiment, said steps (a) and / or (b) are carried out in a culture or culture medium that allows the growth of a sample comprising eukaryotic cells (preferably specific for the growth of said eukaryotic cells).

[0075] More specifically, the culture conditions (including time and cell culture medium) of step (a) or (b) are suitable for the eukaryotic cells to be labeled, detected or targeted. The cell culture medium may be supplemented with any compound to enhance or stimulate cell multiplication and / or the assimilation of the modified monosaccharide compound of formula (I) on the cell surface or cell membrane.

[0076] According to a specific embodiment, the duration of step (a) allows the incorporation of at least one monosaccharide compound of formula (I) or a precursor thereof into the membrane of the eukaryotic cell. More specifically, the duration of step (a) is at least the doubling time of the eukaryotic cell to be labeled, detected or targeted. More specifically, the duration of step (a) is less than five times the doubling time of the eukaryotic cell to be labeled, detected or targeted. According to a specific embodiment, the duration of step (a) corresponds to one doubling time or two doubling times of the eukaryotic cell to be labeled, detected or targeted.

[0077] According to a particular embodiment, said steps (a) and / or (b) are performed with reactants and / or catalysts to produce a reaction of said first reactive group with said second reactive group.

[0078] It is noteworthy that the monosaccharide compound of formula (I) or its precursor exhibits low or no toxicity to cells as shown in the Examples, and therefore its dosage can vary over a wide range. Such an amount will be determined by those skilled in the art so that the amount is sufficient to label, identify or detect eukaryotic cells.

[0079] The method can be implemented with any sample, typically a biological sample of the experimenter, for example, a fluid, such as a sample of blood, plasma, serum, urine, cerebrospinal fluid or a sample from the tissue of the experimenter or its part. The present invention can be implemented with a sample from any experimenter, including any human patient suffering from or suspected of having cancer. The method is usually carried out to a sample of blood, serum or plasma or a sample derived from blood, serum or plasma, such as a pretreated blood sample. The sample can be processed (for example, diluted, concentrated, separated, partially purified, frozen, etc.) before being used in the present invention. According to a specific embodiment, each sample used in the step (a) of the method comprises a cell mass or preferably a single cell, preferably obtained by cell sorting, particularly obtained by flow cytometry. When the method is implemented in vivo, the method can be implemented to the whole body or a part thereof of the experimenter. In this case, the monosaccharide compound of formula (I) or its precursor and the optional compound with the first reactive group can be administered enterally (including orally) or parenterally (including intravenously or intramuscularly).

[0080] For detecting coupled eukaryotic cells from a multicellular organism, the method further comprises a step c) comprising detecting the binding of the compound of step (b) to the monosaccharide of step (a).

[0081] Advantageously, the present invention comprises an additional step (c) of detecting eukaryotic cells to detect whether said eukaryotic cells are coupled to the compound carrying the first reactive group of step (b) and / or immobilizing said eukaryotic cells coupled to said compound carrying the first reactive group on a solid matrix, wherein said compound carrying the first reactive group is a moiety or molecule comprising a detectable substance or capable of reacting or binding with a detectable substance, or preferably said compound carrying the first reactive group is a first molecule capable of reacting or binding with a second molecule and / or binding to a solid matrix, preferably said second molecule comprising a detectable substance and / or said second molecule is bound or capable of binding to said solid matrix.

[0082] Thus, the present invention enables labeling, numbering or detection, and concentration and / or separation of eukaryotic cells from multicellular organisms, optionally immobilized on a solid support; in particular a solid support consisting of magnetic beads carrying said first reactive groups.

[0083] More particularly, the compound carrying a first reactive group is a first molecule capable of reacting or binding with a second molecule and / or binding to a solid matrix, preferably, the second molecule comprises a detectable substance, and the method comprises a step c) of detecting a eukaryotic cell to detect whether the eukaryotic cell comprises the detectable molecule or part bound to the eukaryotic cell.

[0084] According to the method of the present invention, without labeling or detection, it can be concluded that the sample applied does not contain any eukaryotic cells from a multicellular organism.

[0085] The detection step c) can be performed in a liquid medium or on a solid matrix.

[0086] Preferably, the method for labeling or detecting or targeting eukaryotic cells from a multicellular organism is an in vitro method.

[0087] According to certain embodiments, the method of the present invention may further comprise one or more washing steps.

[0088] According to a specific embodiment, the method according to the invention can be performed simultaneously on one or more samples using, for example, a microplate. The microplate typically has 6, 12, 24, 48, 96, 384 or 1536 sample wells. According to said specific embodiment, each sample well used in step (a) of the method preferably contains a cell population or preferably a single cell, more preferably obtained by cell sorting, in particular by flow cytometry.

[0089] Therefore, a further object of the present invention is the in vitro use of at least one modified monosaccharide compound of formula (I) or a precursor thereof (preferably formula (I')), preferably with a compound comprising a first reactive group, and optionally with a second molecule and / or bound to a solid matrix, for labeling or detecting eukaryotic cells from multicellular organisms, preferably, said second molecule comprising a detectable substance.

[0090] A further object of the present invention is a kit for carrying out the method of labeling or detecting eukaryotic cells from a multicellular organism as defined herein, comprising:

[0091] - a modified monosaccharide compound of formula (I), preferably formula (I'), or a precursor thereof, as defined above, and

[0092] - a compound carrying a first reactive group, as defined above.

[0093] According to a specific embodiment, the kit may further comprise a second molecule and / or a solid matrix as defined above, preferably, the second molecule or solid matrix comprises a detectable substance, and the compound carrying a first reactive group is a first molecule capable of reacting or binding with the second molecule and / or binding to the solid matrix.

[0094] A further object is the use of a kit as defined above for carrying out a method for labelling or detecting eukaryotic cells from a multicellular organism as defined herein.

[0095] According to a specific embodiment, the eukaryotic cell from the multicellular organism is a cell susceptible to cancer or tumor cells.Thus, the method and kit according to the present invention can be used to identify cancer or tumor cells by detecting the marker.

[0096] Methods for identifying or isolating cancer cells or diagnosing cancer

[0097] The present invention further relates to a method, preferably an in vitro or ex vivo method, for identifying or isolating cancer cells or diagnosing cancer in a subject, the method comprising carrying out a step for labeling or detecting eukaryotic cells as defined herein in the subject or in a biological sample from the subject. In a preferred embodiment, the method for identifying or isolating cancer cells or diagnosing cancer in a subject further comprises the step of detecting the label and optionally comparing the label to a reference level.

[0098] The biological sample from the subject is as defined above, and preferably, the sample is a sample from a human patient suffering from or suspected of suffering from cancer. According to a specific embodiment, each sample used in step (a) comprises a population of cells or preferably single cells, preferably obtained by cell sorting, in particular by flow cytometry.

[0099] More particularly said sample is suspected of comprising cancer cells.

[0100] Examples of such samples include fluids, such as blood, plasma, saliva, urine and semen samples, as well as biopsy, organ, tissue or cell samples.The sample may be processed before use.

[0101] Cancer cells identified or separated according to the present invention can be of any type. They may be from solid tumors or hematopoietic cancers. Cancer cells include circulating or non-circulating tumor cells. Circulating tumor cells (CTCs) are cells that are shed from the primary tumor into the vascular system or lymphatic system and carried throughout the body in the blood circulation. CTCs constitute seeds for the subsequent growth of other tumors (metastasis) in distant organs, which is the mechanism causing the vast majority of cancer-related deaths.

[0102] The detection and analysis of cancer cells according to the present invention can help early patient prognosis and determine suitable customized treatment. The ability to monitor disease progression over time can promote appropriate modifications to patient treatment, which is likely to improve their prognosis and quality of life. In the case of detecting and analyzing circulating tumor cells, the method can allow early detection of cancer, particularly metastatic cancer. In this regard, the sample according to the present invention is blood. Blood testing is both simple and safe, and multiple samples can be collected over time. An important aspect of the ability to predict future disease progression is to eliminate (at least temporarily) the need for surgery when repeated CTC counts are low and do not increase; the obvious benefits of avoiding surgery include avoiding the risks associated with the innate tumorigenicity of cancer surgery. For this reason, the technology of the sensitivity and repeatability required for detecting CTCs in patients with metastatic disease as the present invention has aroused great interest.

[0103] As used herein, the expression "detecting a marker" can include visualizing or detecting the presence of a marker in a multicellular organism or can also include measuring such a marker. Measurement of such a marker (e.g., fluorescence) allows for the detection, identification, or isolation of cancer cells, optionally by comparing the marker to a reference level.

[0104] It has been found herein that the modified monosaccharide compounds of formula (I) or their precursors, preferably formula (I'), are assimilated differently in cancer cells than in non-cancerous cells, and more particularly at a higher level compared to non-cancerous cells (e.g., reference levels or control samples). As shown in the Examples, the fluorescence intensity of cancer cells is higher (e.g., after one or two cell doubling times) than that of non-cancerous cells.

[0105] Therefore, the present invention relates to a method for identifying or isolating cancer cells or for diagnosing cancer in a subject, comprising:

[0106] - performing a method for labeling eukaryotic cells as described herein in a sample from said subject;

[0107] - detecting the marker after performing the labeling method and optionally comparing the marker to a reference level; then

[0108] - Identifying cancer cells or diagnosing cancer based on measurement of the marker.

[0109] The method for identifying or separating cancer cells or diagnosing cancer can advantageously be carried out within a cell cycle time period. The detection of the label is preferably carried out 10 to 40 hours after implementing the labeling method, more preferably 16 to 24, 25 or to 36 hours, and more specifically after implementing step (a) as described in detail above.

[0110] The method may optionally include comparison with a reference level, more specifically a control sample or reference. The reference level can be a marker (e.g., fluorescence) intensity measured in normal cells (e.g., non-cancerous cells) and / or known cancer cells. Preferably, the reference cell is a cell closest to the cell to be studied, preferably from the same cell line, the same organ, and / or the same type of cell. The method may include a prior step of providing a tumor sample and a histologically matched normal tissue from the subject.

[0111] According to a specific embodiment, when the measured value of the marker of the sample is higher than the measured value of the marker of the control sample, which is a non-cancerous sample, it is identified as a cancer cell or diagnosed as cancer. "Higher measured value" refers to a marker ratio of the sample relative to the non-cancerous sample that is greater than 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or 3.0. More specifically, the marker ratio of the sample relative to the non-cancerous sample is greater than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or 3.0.

[0112] According to a specific embodiment, the cancer to be diagnosed is selected from the group consisting of rectal cancer, colorectal cancer, stomach cancer, head and neck cancer, thyroid cancer, cervical cancer, uterine cancer, breast cancer, ovarian cancer, brain cancer, lung cancer, skin cancer, bladder cancer, blood cancer, kidney cancer, liver cancer, prostate cancer, multiple myeloma and endometrial cancer. More specifically, the cancer to be diagnosed is selected from the group consisting of bladder cancer, blood cancer, skin cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, lung cancer, muscle cancer, lymphocyte cancer, prostate cancer, stomach cancer and breast cancer. According to a more specific embodiment, the cancer to be diagnosed is selected from the group consisting of bladder cancer, blood cancer, colon cancer, stomach cancer, breast cancer, lung cancer, skin cancer and pancreatic cancer.

[0113] According to a specific embodiment, the cancer cells to be identified or isolated are cancer cells derived from the above-mentioned cancers.

[0114] Pharmaceutical composition and use thereof

[0115] The present invention relates to a composition comprising a eukaryotic cell having on its surface at least one modified monosaccharide compound of formula (I) as defined above or a precursor thereof, preferably formula (I'), which modified monosaccharide compound of formula (I) or a precursor thereof may or may not be operably linked to an anticancer drug or to particles comprising at least one anticancer drug.

[0116] In a particular aspect, cells having at least one modified monosaccharide compound of formula (I) as defined above or a precursor thereof present on their surface (which is operably linked or not to an anticancer drug or a particle comprising at least one anticancer drug) can be prepared according to the above-mentioned method of the present invention.

[0117] The composition according to the present invention can be prepared by a method comprising the following steps:

[0118] a) contacting a composition comprising eukaryotic cells from a multicellular organism with at least one modified monosaccharide compound as defined above or a precursor thereof;

[0119] b) contacting the composition of step (a) with a compound bearing a first reactive group and optionally comprising an operably linked anticancer drug or particles comprising at least one anticancer drug, optionally in the presence of copper.

[0120] The steps are similar to those described above. Step a) allows the cell to present at least one modified monosaccharide compound of formula (I) or a precursor thereof on its surface or membrane. Step b) allows a click chemistry reaction to occur between the first reactive group of the compound and the second reactive group X of at least one modified monosaccharide compound of formula (I) or a precursor thereof, preferably formula (I').

[0121] According to one embodiment, the compound having a first reactive group may also contain or may be an operably linked anticancer drug or a particle comprising at least one anticancer drug linked to said compound, and step (b) allows providing a eukaryotic cell from a multicellular organism coupled to the anticancer drug or the particle comprising the anticancer drug.

[0122] Thus, the method allows the preparation of a conjugate by a click chemistry reaction as detailed above, wherein a modified monosaccharide compound of formula (I) or a precursor thereof of formula (I') is operably linked to an anticancer drug or a particle comprising the anticancer drug, and the conjugate is present on the surface of a eukaryotic cell. In a specific embodiment, the conjugate is prepared by a click chemistry reaction between an anticancer drug comprising an alkynyl group or a particle having an alkynyl group present on its surface and a modified monosaccharide compound of formula (I) comprising an azide group or a precursor thereof of formula (I').

[0123] According to another embodiment, the compound with the first reactive group is a first ligand (or more specifically a first binding protein) that can react or bind to a second ligand (or more specifically a second binding protein) as an anticancer agent. According to such an embodiment, the method allows the preparation of a conjugate by a click chemistry reaction as detailed above, wherein the modified monosaccharide compound of formula (I) or its precursor formula (I') is connected to a first ligand that can react or bind to a second ligand as an anticancer agent, and the conjugate is present on the surface of a eukaryotic cell. In a specific embodiment, the conjugate is prepared by a click chemistry reaction as detailed above.

[0124] As used herein, "anticancer drug" corresponds to any drug currently used for cancer treatment, such as an antitumor drug. In a preferred embodiment, the anticancer drug is selected from chemotherapeutic agents, anticancer antibodies, hormone therapy, immunotherapy and kinase inhibitors.

[0125] The term "operably linked anticancer drug" refers to an anticancer drug that is linked, preferably covalently linked, while being able to exhibit its therapeutic effect.

[0126] The particles comprising at least one anticancer drug are particles containing the anticancer drug, preferably nanoparticles, having a first reactive group as defined above. For example, the particles can be bicyclo[6.1.0]nonyne-modified glycol chitosan nanoparticles (BCN-CNPs). As is well known, CNPs can encapsulate carious drugs with high compatibility and are widely used in drug delivery.

[0127] Chemotherapy may include topoisomerase I or II inhibitors, DNA cross-linking agents, DNA alkylating agents, antimetabolites, and / or mitotic spindle inhibitors.

[0128] Inhibitors of topoisomerases I and / or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, indolesin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idanrubicine, and mitoxantrone. Inhibitors of topoisomerases I and II include, but are not limited to, indolesin.

[0129] DNA cross-linking agents include, but are not limited to, cisplatin, carboplatin, and oxaliplatin.

[0130] Antimetabolites block the enzymes responsible for nucleic acid synthesis or are incorporated into DNA, thereby generating an incorrect genetic code and leading to apoptosis. Non-exhaustive examples include, but are not limited to, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, more particularly methotrexate, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, 5-fluorouracil, gemcitabine, and capecitabine.

[0131] Alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, metal salts, and triazenes. Non-exhaustive examples include uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, fotemustine, cisplatin, carboplatin, oxaliplatin, thiotepa, streptozotocin, dacarbazine, and temozolomide.

[0132] Mitotic spindle inhibitors include, but are not limited to, paclitaxel, docetaxel, vinorelbine, larotaxel (also known as XRP9881; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol-Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also known as IDN 5109, BAY 59-8862, or SB-T-101131; Bristol-Meyer-Squibb), RPR 109881A (Bristol-Meyer-Squibb), RPR116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel (also known as CT-2103, PPX, paclitaxel poliglumex, paclitaxel polyglutamate, or Xyotax TM ), (also known as Nab-paclitaxel; ABRAXISBIOSCIENCE), Tesetaxel (also known as DJ-927), IDN 5390 (INDENA), Taxoprexin (also known as docosahexanoic acid-paclitaxel; PROTARGA), DHA-paclitaxel (also known as ) and MAC-321 (WYETH). See also the review by Henneenfent & Govindan (2006, Annals of Oncology, 17, 735-749).

[0133] Immune checkpoint inhibitors may be selected from anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) therapy such as ipilimumab, PD-1 (programmed cell death protein 1) inhibitors such as nivolumab, pembrolizumab or BGB-A317, PDL1 (programmed cell death ligand) inhibitors such as atezolizumab, avelumab or durvalumab, LAG-3 (lymphocyte activation gene 3) inhibitors such as BMS-986016, TIM-3 (T cell immunoglobulin and mucin domain-containing-3) inhibitors, TIGIT (T cell immunoreceptor with Ig and ITIM domains) inhibitors, BLTA (B and T lymphocyte attenuator) inhibitors, IDO1 inhibitors such as epacadostat, or a combination thereof.

[0134] Hormonal therapies include, for example, Tamoxifen, Fareston, Arimidex, Aromasin, Femara, Zoladex / Lupron, Megace, and Halotestin.

[0135] The eukaryotic cells from the multicellular organisms of the compositions of the present invention are preferably isolated non-cancerous cells. The cells are preferably isolated pluripotent stem cells. The cells are preferably mesenchymal stem cells (MSCs). MSCs are present throughout the body, preferably in adipose tissue, bone marrow, tissues supporting organs, and also in bone, cartilage, and muscle. The eukaryotic cells of the present invention may be T cells.

[0136] The cells are allogeneic cells or preferably autologous cells (ie, derived from the subject or patient himself).

[0137] According to a specific embodiment, the composition comprises MSCs having at least one modified monosaccharide compound of formula (I) as defined above, or a precursor thereof, present on their surface. Such compositions can be used, for example, to identify cancer cells or tumors or diagnose cancer by using the properties of the monosaccharide to be detected directly or indirectly by the compound comprising the first reactive group, as described in detail above. Such compositions can also be used therapeutically to monitor hematopoietic transplantation.

[0138] According to another specific embodiment, the composition comprises MSCs having on their surface at least one modified monosaccharide compound of formula (I) as defined above, or a precursor thereof, operatively linked to an anticancer drug or to particles comprising at least one anticancer drug and linked to said compound. Such a composition may also be used in therapy, in particular in the treatment of cancer.

[0139] The composition of the present invention is preferably a pharmaceutical composition.

[0140] In addition to the cells of the presence of anticancer drugs as described in detail above, the pharmaceutical composition considered herein also includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is intended to include any carrier (e.g., support, material, solvent, etc.) that does not interfere with the effectiveness of the biological activity of the cells and is nontoxic to the host to which it is applied. For example, for parenteral administration, the active compound can be formulated into a unit dosage form for injection in a vehicle, such as saline, dextrose solution, serum albumin, and Ringer's solution. The pharmaceutical composition can be formulated into a solution in a pharmaceutically compatible solvent, or into an emulsion, suspension, or dispersion in a suitable pharmaceutical solvent or vehicle, or into a pill, tablet, or capsule containing a solid vehicle in a manner known in the art. Suitable preparations for parenteral administration conveniently include sterile oily or aqueous preparations of active ingredient, which are preferably isotonic with the blood of the recipient.

[0141] The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical composition is advantageously administered by injection or intravenous infusion of a suitable sterile solution. Safe and effective methods of administering most of these chemotherapeutic agents are known to those skilled in the art. In addition, methods of administering them are described in standard literature.

[0142] The pharmaceutical composition of the present invention can be used for cancer therapy, more particularly for cancer cell therapy.

[0143] A preferred embodiment of the present invention is a pharmaceutical composition as defined herein for use in the treatment of cancer as defined above.

[0144] A further preferred embodiment is a method of treating cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition as defined herein.

[0145] A further preferred embodiment is the use of a pharmaceutical composition as defined herein for the preparation of a medicament for the treatment of cancer.

[0146] Imaging and diagnostics

[0147] As detailed above, the modified monosaccharide compound of formula (I) or its precursor formula (I') is suitable for forming a detectable entity with a label, preferably a fluorescent label, in particular by a click chemistry reaction.

[0148] Therefore, the present invention relates to the use of modified monosaccharide compounds of formula (I) disclosed herein or its precursor formula (I') as a research tool for detecting eukaryotic cells from multicellular organisms, and more specifically to the use for identifying or isolating cancer cells. The present invention further relates to modified monosaccharide compounds of formula (I) disclosed herein or its precursor formula (I') for use in medical imaging or diagnosis, preferably for diagnosing cancer.

[0149] Further aspects and advantages of the present invention will be described in the following examples, which are to be regarded as illustrative rather than limiting. Example

[0150] Example 1 :Synthesis of compounds

[0151] Materials and methods:

[0152] Thin layer chromatography was performed on a Merck 60F254 with detection by UV and / or by charring with sulfuric acid, KMnO4 or phosphomolybdic acid solutions. Silica gel 60 40-63 mm was used for flash column chromatography.

[0153] NMR spectra were acquired on a Bruker Avance 300 or 500 MHz spectrometer using residual protonated solvent as an internal standard. Chemical shifts δ are given in parts per million (ppm), and coupling constants are reported in Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), doublet of a doublet (dd), or doublet of a doublet of a doublet (ddd). Splitting patterns that could not be interpreted or easily visualized were designated as multiplets (m).

[0154] Mass spectra were acquired on a Waters LCT Premier XE (ToF) with electrospray ionization in positive (ESI+) or negative (ESI-) detection mode.

[0155] IR-FT spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Characteristic absorption is expressed in cm -1 Report to the unit.

[0156] Specific optical rotation was measured at 20°C with an Anton Paar MCP 300 polarimeter in a 10-cm cell at 20°C and 589 nm.

[0157] All biological and chemical reagents were of analytical or cell culture grade, obtained from commercial sources, and used without further purification.

[0158] Ara-N3 and its precursor VI have been synthesized according to the following steps:

[0159]

[0160] where R 1 、R 2 and R 3 It is a methyl group.

[0161] 2:3,4:5-Diisopropylidene-D-arabinose O-methyloxime (Compound II)

[0162] To a solution of D-(-)-arabinose (4.00 g, 26.6 mmol, 1.0 eq.) in anhydrous pyridine (90 mL) was added methoxyamine hydrochloride (2.72 g, 32.0 mmol, 1.2 eq.) and the mixture was stirred at room temperature for 15 hours. The solvent was removed under reduced pressure and the residue was co-evaporated three times with toluene. The residue was resuspended in 2,2-dimethoxypropane (100 mL) and 7-toluenesulfonic acid (1.01 g, 5.33 mmol, 0.2 eq.) was added and the suspension was heated to reflux for 4 hours and then stirred at room temperature for another 15 hours. The reaction mixture was heated to reflux for 4 hours and then stirred at room temperature for 15 hours. At 4 DEG C, 80 DEG C of 4-diisopropyl-D-arabinose O-methyl oxime (IIE / IIZ) is added into the 4-nitro-2-nitro-1-oxo-2-nitro-2-oxo-3-nitro-3-oxo-4-nitro-3-oxo-3 ...

[0163] Isomers (IIE):

[0164] Rf(CH2Cl2 / MTBE 98:2): 0.35.

[0165] IR(cm -1 ): 2987,2939,2900,2821,1631,1456,1381,1371,1241,1212,1150,1065,1038,887,842.

[0166] 1 H-NMR (500MHz, CDCl3) δ: 7.35 (d, 1H, J 1,2 6.3Hz,Hl);4.46(dd,1H,J 2,3 7.1,J 1,2 6.3Hz,H-2);4.13(ddd,1H,J 3,4 6.9,J 4,5a 6.1,J 4,5b 4.8Hz,H-4); 4.08(dd,1H,J 5a,5b 8.5,J 4,5a 6.1Hz,H-5a); 3.97(d,1H,J 2,3 7.1,J 3,46.9Hz,H-3);3.94(dd,1H,J 5a,5b 8.5,J 4,5b 4.8Hz,H-5b); 3.85(s,3H,CH3-O); 1.40(s,3H,CH3-C); 1.38(s,6H,2CH3-C); 1.32(s,3H,CH3-C).

[0167] 13 C-NMR (125MHz, CDCl3) δ: 147.8 (Cl); 110.8 (C-6); 110.0 (C-7); 79.4 (C-3); 76. 7(C-2); 76.6(C-4); 67.1(C-5); 62.1(CH3-O); 27.1, 27.0, 26.9, 25.4(4CH3-C).

[0168] HRMS(ESI + ): [M+H] + (C 12 H 22 NO5 + ) Calculated value m / z: 260.1492, measured value: 260.1502.

[0169] 2:3-Isopropylidene-D-arabinose O-methyloxime (Compound III)

[0170] A solution of 2:3,4:5-diisopropylidene-D-arabinose O-methyloxime (II) (IIE / IIZ) and an impurity (1.50 g) in 80% (v / v) aqueous acetic acid (30 mL) was heated to 40°C on a rotary evaporator at 200 mbar. After 2.5 hours, the solvent was removed under reduced pressure, and the residue was co-evaporated with toluene. Flash column chromatography on silica gel (cyclohexane / ethyl acetate 1:1) afforded a mixture of isomers of 2:3-diisopropylidene-D-arabinose O-methyloxime (IIIE / IIIZ) (NMR ratio 4:1, 876 mg, 58% over 3 steps) as a colorless oil. The NMR purity was greater than 95%.

[0171] Rf (cyclohexane / ethyl acetate 1:1): 0.24.

[0172] IR(cm -1 ):3409,2939,1373,1216,1040,885.

[0173] HRMS(ESI + ): [M+H] + (C9H 18 NO5 +) Calculated value m / z: 220.1179, measured value: 220.1184.

[0174] Isomer (IIIE):

[0175] 1 H-NMR (500MHz, CDCl3) δ: 7.44 (d, 1H, J 1,2 5.5Hz,Hl);4.56(dd,1H,J 2,3 7.4,J 1,2 5.5Hz,H-2);4.07(dd,1H,J 2,3 7.4,J 3,4 5.6Hz,H-3);4.13(ddd,1H,J 3,4 5.6,J 4,5 5.1,J 4,5 4.7Hz,H-4); 3.84(s,3H,CH3-O); 3.72-3.68(m,2H,2H-5); 1.42(s,3H,CH3-C); 1.38(s,3H,CH3-C).

[0176] 13 C-NMR (125MHz, CDCl3) δ: 149.1 (C-1); 110.3 (C-6); 79.4 (C-3); 75.0 (C-2); 71.6 (C-4); 63.4 (C-5); 62.2 (CH3-O); 26.9, 26.7 (2CH3-C).

[0177] Isomer (IIIZ):

[0178] 1 H-NMR (500MHz, CDCl3) δ: 6.86 (d, 1H, J 1,2 5.9Hz,Hl);4.95(dd,1H,J 2,3 7.7,J 1,2 5.9Hz,H-2); 3.92(s,3H,CH3-O); 3.87(dd,1H,J 2,3 7.7,J 3,4 6.9Hz, H-3); 3.82-3.75 (m, 2H, H-4, H-5a); 3.72-3.68 (m, 1H, H-5b); 1.40 (2s, 6H, 2CH3-C).

[0179] 13C-NMR (125MHz, CDCl3) δ: 151.0 (C-1); 110.9 (C-6); 80.4 (C-3); 72.9 (C-2); 72.7 (C-4); 63.5 (C-5); 62.8 (CH3-O); 27.0, 26.5 (2CH3-C).

[0180] 2:3-Isopropylidene-5-O-methylsulfonyl-D-arabinose O-methyloxime (Compound IV)

[0181] To a solution of 2:3-isopropylidene-D-arabinose O-methyloxime (III) (IIIE / IIIZ) (10 mg, 0.46 mmol, 10 eq.) in anhydrous pyridine (2.0 mL) at -20°C was added methanesulfonyl chloride (0.10 mL, 1.37 mmol, 3.0 eq.). The reaction mixture was stirred at -20°C for 1.5 hours. The reaction was quenched with CHOH (0.3 mL), and the solvent was removed in vacuo. The resulting residue was purified by silica gel flash column chromatography (cyclohexane / ethyl acetate 6:4) to afford a mixture of 2:3-isopropylidene-5-O-methylsulfonyl-D-arabinose O-methyloxime (IVE / IVZ) (NMR ratio 4:1, 110 mg, 81%) as a colorless oil. An aliquot of the pure (IVE) isomer was obtained and characterized by flash column chromatography (dichloromethane / ether 9:1). The NMR purity was greater than 95%.

[0182] Rf (cyclohexane / ethyl acetate 6:4): 0.24.

[0183] IR(cm -1 ): 3500,2989,2941,2824,1631,1458,1350,1215,1170,1067,1033,959,887,863,833.

[0184] HRMS(ESI + ): [M+H] + (C 10 H 20 NO7S + ) Calculated value m / z: 298.0955, measured value: 298.0947.

[0185] Isomers (IVE):

[0186] Rf (cyclohexane / ethyl acetate 6:4): 0.20.

[0187] 1 H-NMR (500MHz, CDCl3) δ: 7.42 (d, 1H, J 1,25.6Hz,H-l);4.56(dd,1H,J 2,3 6.8,J 1,2 5.6Hz,H-2);4.41(dd,1H,J 5a,5b 11.0,J 4,5a 2.7Hz,H-5a);4.28(dd,1H,J 5a,5b 11.0,J 4,5b 5.7Hz,H-5b);4.03(ddd,1H,J 3,4 7.0,J 4,5b 5.7,J 4,5a 2.7Hz,H-4);4.01(dd,1H,J 2,3 6.8,J 3,4 7.0Hz,H-3);3.85(s,3H,CH3-O);3.06(s,3H,CH3-S);1.41(s,3H,CH3-C);1.39(s,3H,CH3-C)。

[0188] 13 C-NMR(125MHz,CDCl3)δ:148.2(C-1);110.9(C-6);77.9(C-3);76.2(C-2);70.9(C-5);70.8(C-4);62.3(CH3-O);37.8(CH3-S);27.0,26.9(2CH3-C)。

[0189] Isomer (IVZ):

[0190] 1 H-NMR(300MHz,CDCl3)δ:6.87(d,1H,J 1,2 5.9Hz,H-l);4.96(dd,1H,J 2,3 7.3,J 1,2 5.9Hz,H-2);4.45(dd,1H,J 5a,5b 11.4,J 4,5a 2.4Hz,H-5a);4.29(dd,1H,J 5a,5b 11.4,J 4,5b 7.9Hz,H-5b);3.98(ddd,1H,J 4,5b 7.9,J 4,3 7.5,J 4,5a 2.4Hz,H-4);3.93(s,3H,CH3-O);3.84(dd,1H,J 3,4 7.5,J 3,27.3Hz,H-3); 3.06(s,3H,CH3-S); 1.40(s,3H,CH3-C); 1.39(s,3H,CH3-C).

[0191] 13 C-NMR (75MHz, CDCl3) δ: 150.7 (C-1); 111.2 (C-6); 79.1 (C-3); 72.9 (C-2); 71.3 (C-5); 70.9 (C-4); 62.9 (CH3-O); 37.9 (CH3-S); 27.0, 26.6 (2CH3-C).

[0192] 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloxime (Compound V):

[0193] To a solution of (IVE / IVZ) (810 mg, 2.72 mmol, 1.0 eq.) in N,N-dimethylformamide (30.0 mL, 0.10 M) was added sodium azide (531 mg, 8.17 mmol, 3.0 eq.), and the reaction mixture was heated at 80°C for 15 hours. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography (cyclohexane / ethyl acetate 9:1) to yield a mixture of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose O-methyloximes (VE / VZ) (NMR ratio 7:3, 637 mg, 96%) as a pale yellow oil. A portion of (VE) was isolated by flash column chromatography (dichloromethane / MTBE 97:3) for characterization. The NMR purity was greater than 95%.

[0194] Rf (cyclohexane / ethyl acetate 8:2): 0.30.

[0195] IR(cm -1 ):3458,2989,2939,2823,2100,1630,1443,1373,1213,1164,1066,1036,885,865.

[0196] HRMS(ESI + ): [M+H] + (C9H 17 N4O4 + ) Calculated value m / z: 245.1245, measured value: 245.1250.

[0197] Isomers (VE):

[0198] Rf (dichloromethane / MTBE 97:3): 0.23.

[0199] 1H-NMR(500MHz,CDCl3)δ:7.42(d,1H,J 1,2 5.8Hz,H-l);4.54(dd,1H,J 2,3 7.2,J 1,2 5.8Hz,H-2);3.98(dd,1H,J 2,3 7.2,J 3,4 6.4Hz,H-3);3.92(dddd,1H,J 3,4 6.4,J 4,5b 6.2,J 4,OH 3.9,J 4,5a 3.8Hz,H-4);3.85(s,3H,CH3-O);3.46(dd,1H,J 5a,5b 12.5,J 4,5a 3.8Hz,H-5a);3.42(dd,1H,J 5a,5b 12.5,J 4,5b 6.2Hz,H-5b);2.61(d,1H,J 4,OH 3.9Hz,OH);1.41(s,3H,CH3-C);1.39(s,3H,CH3-C)。

[0200] 13 C-NMR(125MHz,CDCl3)δ:148.4(C-l);110.6(C-6);78.8(C-3);75.8(C-2);71.5(C-4);62.3(CH3-O);53.7(C-5);27.0,26.8(2CH3-C)。

[0201] Isomer (VZ):

[0202] 1 H-NMR(500MHz,CDCl3)δ:6.86(d,1H,J 1,2 6.1Hz,H-l);4.94(dd,1H,J 2,3 7.2,J 1,2 6.1Hz,H-2);3.93(s,3H,CH3-O);3.87(ddd,1H,J 3,4 7.5,J 4,5b 6.4,J 4,5a 2.8Hz,H-4);3.82(dd,1H,J 3,4 7.5,J 2,3 7.2Hz,H-3);3.47(dd,1H,J 5a,5b 12.8,J 4,5a2.8Hz,H-5a);3.39(dd,1H,J 5a,5b 12.8,J 4,5b 6.4Hz,H-5b); 1.40(s,3H,CH3-C); 1.38(s,3H,CH3-C).

[0203] 13 C-NMR (75MHz, CDCl3) δ: 150.8 (Cl); 111.0 (C-6); 80.0 (C-3); 72.9 (C-2); 72.5 (C-4); 62.8 (CH3-0); 53.5 (C-5); 26.9, 26.5 (2CH3-C).

[0204] 5-Azido-5-deoxy-2:3-isopropylidene-D-arabinose (Compound VI)

[0205] To a solution of (VE / VZ) (820 mg, 3.36 mmol, 1.0 eq.) in 80% (v / v) aqueous acetic acid (120 mL) was added formaldehyde (0.8 mL) and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and co-evaporated with toluene to ensure complete elimination of acetic acid. This afforded the crude compound 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (VI) (682 mg).

[0206] Colorless oil

[0207] Rf (cyclohexane / ethyl acetate 7:3): 0.56.

[0208] IR(cm -1 ):3408,2988,2936,2100,1733,1440,1373,1238,1213,1164,1063,863.

[0209] 1 H-NMR (500MHz, CDCl3) δ: 9.79 (d, 1H, J 1,2 1.2Hz,Hl);4.41(dd,1H,J 2,3 6.4,J 1,2 1.2Hz,H-2);4.04(dd,1H,J 2,3 6.4,J 3,4 6.1Hz,H-3);3.90(ddd,1H,J 4,5b 6.4,J 3,4 6.1,J 4,5a 3.4Hz,H-4); 3.51(dd,1H,J 5a,5b 12.8,J 4,5a3.4Hz,H-5a); 3.43(dd,1H,J 5a,5b 12.8,J 4,5b 6.4Hz,H-5b); 1.47(s,3H,CH3-C); 1.37(s,3H,CH3-C).

[0210] HRMS(ESI + ):[2M+Na] + (C 16 H 26 N6NaO8 + ) Calculated value m / z: 453.1704, measured value: 453.1726.

[0211] 5-Azido-5-deoxy-D-arabinofuranosyl (Compound VII, Ara-N3)

[0212] To a solution of 5-azido-5-deoxy-2:3-isopropylidene-D-arabinose (VI) (100 mg) in a mixture of CH2Cl2 / H2O (20:1, 21 mL) was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated, and the crude residue was resuspended in water and lyophilized. After flash column chromatography on silica gel (dichloromethane / methanol 92:8), compound 5-azido-5-deoxy-D-arabinofuranosyl or Ara-N3(VII) (50 mg, 58%, 2 steps from compound (V)) was obtained as a colorless oil with a mixture of α / β anomers (NMR ratio 55:45). The NMR purity was greater than 95%.

[0213] Rf (dichloromethane / methanol 92:8): 0.28.

[0214] IR(cm -1 ):3367,2106,1281,1040.

[0215] HRMS(ESI + ): [M+H-N2] + (C5H10NO4 + ) Calculated value m / z: 148.0604, measured value: 148.0610.

[0216] Anomer α(VIIα):

[0217] 1 H-NMR(500MHz,D2O)δ: 5.24(d,1H,J 1,2 2.9Hz,Hl);4.17(ddd,1H,J 3,4 6.4,J4 ,5b 5.8,J4,5a 3.5Hz,H-4);4.01(dd,1H,J 2,3 4.6,J 1,2 2.9Hz,H-2);3.97(dd,1H,J 3,4 6.4,J 3,2 4.6Hz,H-3);3.64(dd,1H,J 5a,5b 13.6,J 4,5a 3.5Hz,H-5a);3.44(dd,1H,J 5a,5b 13.6,J 4,5b 5.8Hz,H-5b).

[0218] 13 C-NMR (125MHz, D2O) δ: 101.0 (Cl); 81.3 (C-4); 81.2 (C-2); 76.3 (C-3); 51.5 (C-5).

[0219] Anomer β (VIIβ):

[0220] 1 H-NMR(500MHz,D2O)δ: 5.28(br d,1H,J 1,2 3.1Hz, Hl); 4.10-4.05 (m, 2H, H-2, H-3); 3.89 (ddd, 1H, J 3,4 7.1,J 4,5b 6.5,J 4,5a 3.5Hz,H-4); 3.59(dd,1H,J 5a,5b 13.3,J 4,5a 3.5Hz,H-5a);3.42(dd,1H,J 5a,5b 13.3,J 4,5b 6.5Hz,H-5b).

[0221] 13 C-NMR (125MHz, D2O) δ: 95.2 (Cl); 79.6 (C-4); 75.8 (C-2); 74.7 (C-3); 52.6 (C-5).

[0222] Example 2 : Labeling of non-tumor and tumor eukaryotic cells in the bladder

[0223] Materials and methods

[0224] Cell culture:

[0225] Human urothelial cells (SV-HUC-1) and human bladder carcinoma (T24) were purchased from ATCC (Manassas, Van USA). These cell lines were grown in RPMI 1640 containing glutamine 1 medium (Lonza Biotechnology) supplemented with 10% fetal bovine serum (FBS) (VWR International SAS). The medium was changed every two days. Cells were passaged using Tryp-LE express1x (Gibco). Cell viability was estimated using the trypan blue exclusion assay.

[0226] Cells exposed to Ara-N3 probe:

[0227] SV-HUC-1 and T24 cells were seeded in 24-well plates at a density of 5*10 4 cells / well. The cells were then incubated at 37°C, 5% CO2 for 24 hours. Thereafter, the culture medium in each well was discarded, and the adherent cells were washed twice with phosphate buffered saline (PBS) (Lonza Biowhittaker) and then exposed to the Ara-N3 probe (10 mM). The probe was added to culture media with different FBS concentrations (3% and 10%). The cells were then incubated again at 37°C, 5% CO2 for 24 hours. Control cells were cultured using culture media supplemented with 5% or 10% FBS without the addition of Ara-N3.

[0228] Labeling with anti-biotin antibody:

[0229] Fluorescence incorporation of the Ara-N3 probe was visualized by copper-free click chemistry using sulfo-DBCO-biotin (1 mM) and then labeled with mouse anti-biotin Alexa Fluor 488 antibody conjugate (0.62 mg / ml stock, Jackson ImmunoResearch, diluted 1 / 10). The click reaction was performed as follows: After a 24-hour incubation time, the culture medium was removed and the adherent cells were washed twice with PBS. The cells were detached with Tryp-LE, washed twice with PBS, and centrifuged at 13,000 g for 2 minutes. The cell pellet was resuspended with 10 μL of sulfo-DBCO-biotin and incubated at 37°C in the dark for 30 minutes. Then, the cells were washed twice with PBS, centrifuged, and the cell pellet was resuspended with 10 μL of mouse anti-biotin antibody solution and then incubated at room temperature in the dark. Thereafter, the cells were washed twice with PBS and small cell suspension spots (5 μL) were placed in Adhesion slides (VWR international SAS) were placed in a dark room until dry (20 minutes). The spots were fixed with 4% paraformaldehyde for 20 minutes in the dark at room temperature. The slides were then washed twice with PBS and then recovered with square coverslips (VWR international SAS) using glycerol mounting medium (Dako) and stored in a dark room at 4°C.

[0230] Fluorescence microscopy:

[0231] Fluorescence acquisitions were recorded using an Olympus Microscopy IX83 (Olympus Life Science) equipped with a 60X / 1.3 digital aperture / 1.4 SRI (silicone refractive index) objective and a Hamamatsu oreca flash 4LT camera with a calibrated pixel size of 109 nm / pixel. Excitation light was emitted by an X-CITE 120LED using an AT180 / 30X excitation filter, and the signal was monitored using an AT535 / 40m emission filter. Green light exposure time was 600 milliseconds, and white light exposure time was 340 milliseconds as determined by the DIC method. The size of the acquisition area analyzed by Cellsens Dimension V1.16 software varied depending on the number of cells included in the spot.

[0232] Image processing and fluorescence quantification:

[0233] Images were processed using the Count and Measure module of Cellsens Dimension V1.16 software. Background noise was removed by first defining a ROI on the background of the image and calculating the mean pixel intensity. This value was then subtracted from all pixels in the image. The fluorescence intensity of each cell was determined using a threshold value set at 3030. Any pixels with an intensity below 3030 were not counted.

[0234] result:

[0235] Table 1: Assessment of % Labeled Cells at 24 Hours - FBS Concentration 3%

[0236] <![CDATA[Strength / μm 2 > SV-HUC1% (n=239) T24% (n=226) 0-50000 62.34 2.21 50,000-100,000 2.51 2.65 100,000-150,000 2.09 2.65 150,000-200,000 1.26 5.75 200,000-250,000 1.67 3.98 250,000-300,000 3.77 3.54 300,000-350,000 0.42 2.21 350,000-400,000 0.42 3.98 400,000-450,000 0.84 2.65 450,000-500,000 2.93 3.54 >500,000 21.76 66.81

[0237] Table 2: Assessment of % Labeled Cells at 24 Hours - FBS Concentration 10%

[0238]

[0239]

[0240] The results in Tables 1 and 2 show that the percentage of labeled cells was greater in T24 cells compared to SV-HUC1 cells after 24 hours, demonstrating a higher uptake of the Ara-N3 probe in tumor bladder cells. More specifically, at intensities above 500,000 / μm 2 At 3% FBS, SV-HUC1% was 21.76 and T24% was 66.81, while at 10% FBS, SV-HUC1% was 14.62 and T24% was 79.42. The opposite was true for the percentage of labeled cells obtained at low intensities. The overall distribution of intensities differed between normal and cancer cells.

[0241] Example 3 : Labeling of non-cancerous and cancerous eukaryotic cells

[0242] Materials and methods

[0243] The same experiments as in Example 2 were performed on other cancer cells and corresponding non-cancerous cells. The culture medium was adapted to the cells being tested and supplemented with 10% fetal bovine serum (FBS). The Ara-N3 probe (10 mM) was added to the culture medium containing 10% FBS. The incubation time corresponded to one or two doubling times of the test cells (see the table below).

[0244] The fluorescence signal cancer / non-cancer ratio was calculated based on the fluorescence intensity obtained by testing Ara-N3 on cancer and corresponding non-cancer cells.

[0245] result:

[0246] As shown in the following table (Table 3), the tested cancer cell lines showed higher fluorescence intensity (ratio higher than 1) compared to the corresponding non-cancerous cell lines.

[0247]

[0248]

[0249]

[0250] The same experiments were performed on other human cancer cells, more specifically including the following cells compared to matched non-cancerous cells (Table 4):

[0251] Table 4

[0252] Human cancer cell lines organ disease U87 brain Glioblastoma HUH6 liver Hepatoblastoma HUH7 liver Liver cancer 786-O kidney Renal cell carcinoma NCI-H28 lung Pleural sarcomatoid mesothelioma SW 684 muscle Fibrosarcoma IMR-32 brain neuroblastoma MDA PCa 2b prostate Adenocarcinoma AGS Stomach Gastric adenocarcinoma Kato III Stomach Gastric cancer NCI-N87 Stomach Gastric cancer

[0253] The same results as in Table 3 are expected, ie the tested cancer cell lines have higher fluorescence intensities (especially ratios higher than 1) compared to the corresponding non-cancerous cell lines.

[0254] Example 4-: Comparison of labeling of Ara-N3 and KDO-N3

[0255] Patent application WO 2013 / 107759 describes KDO-N3 (8-azido-3,8-dideoxy-D-manno-octulonic acid) as a useful tool for labeling bacteria.

[0256] Ara-N3 and KDO-N3 were assayed under the same conditions as in Example 2 using other human cell lines: HeLa, Raw, and macrophages.

[0257] The cell line was not labeled with KDO-N3 (no fluorescence intensity was observed) and the cell line was labeled with Ara-N3.

[0258] Example 5: Ara-N3 probe is non-toxic to cells (in vitro / in vivo)

[0259] A-In vitro experiments

[0260] Materials and methods

[0261] The Ara-N3 probe was prepared as detailed above.

[0262] The cell lines (as detailed in the table below) were provided by Inserm / Aquiderm transfer cells. Cell culture and cytotoxicity testing of the cell lines were also performed in the INSERM U1035 laboratory using Aquiderm transfer cells.

[0263] Isolation of peripheral blood mononuclear cells (monocytes / lymphocytes)

[0264] Follow Etablissement du Sang d'Aquitaine established routine, from the peripheral blood from 2 different healthy donors, human mononuclear cells were isolated. The technology used was Ficoll gradient separation. In short, the blood diluted in phosphate buffered saline solution (PBS) was deposited on a sucrose solution (Ficoll, Eurobio) and centrifuged to separate the different blood components. A cell ring consisting of monocytes and lymphocytes was established between Ficoll and the plasma. It was recovered and washed in PBS to count on a Malassez cell.

[0265] Human CD14+ monocytes were isolated from lymphocytes using magnetic beads coupled to anti-CD14 antibodies (Miltenyi Biotech). Monocytes were incubated for 15 minutes in PBS buffer in the presence of beads at a concentration determined based on cell number. The cells were then washed in PBS and placed on a column mounted on a strong magnet. CD14+ cells attached to the anti-CD14 magnetic beads remained attached to the column. After washing, the column was removed from the magnetic support and the cells were separated by washing the column in PBS buffer.

[0266] The number of monocytes was determined by counting on a Malassez cell line.

[0267] Cell culture

[0268] Cells were cultured in an oven at 37°C, 5% CO2 and 95% humidity in different media:

[0269] - For the monocyte / THP1 pair, in RPMI 1640 medium supplemented with 10% decomplemented FCS (fetal calf serum) and the antibiotics penicillin (100 IU / ml) and streptavidin (100 μg / ml).

[0270] - For the Hacat / A431 pair, in DMEM medium supplemented with 10% decomplemented FCS and the antibiotics penicillin (100 IU / ml) and streptavidin (100 μg / ml).

[0271] The culture medium was changed every 2 days throughout the study.

[0272] When the cultures reached 70-80% confluence, adherent cells were detached with 10% trypsin-EDTA. Cell viability was assessed by trypan blue exclusion assay.

[0273] Contact with Ara-N3 probe

[0274] All cells were seeded in culture plates and for MTS proliferation assays were plated at 1*10 4 The density of cells / well in 96-well plates was 5*10 for Annexin V assay / IP assay. 4 Cells were plated at a density of 10 cells / well in 24 wells. The cells were then incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, the medium in all wells was replaced, and cells adhering to the bottom of the wells were either rinsed twice in PBS (Eurobio) or centrifuged and washed twice in PBS to resuspend the cells. The cells were then exposed to the following concentrations of the Ara-N3 probe: 1, 10, 50, and 100 mM. The cells were incubated at 37°C and 5% CO2 for 48 hours.

[0275] Control cells were cultured with supplemented medium without the addition of Ara-N3 probe.

[0276] Annexin V / IP cytotoxicity assay

[0277] This test requires the use of Annexin The V-APC kit (Biolegend) was used to detect cell apoptosis / necrosis. This test utilizes the ability of annexin V to bind to membrane phosphatidylserine (PS) and the intercalation properties of propidium iodide (PI) on DNA.

[0278] Specifically, living cells express PS in the inner layer of their membrane bilayer. Therefore, PS is inaccessible to Annexin V-APC. Similarly, cells maintain the integrity of their DNA away from the nucleus, making it inaccessible to IP. When cells enter apoptosis (programmed cell death), a series of events occurs, including the inversion of the cytoplasmic membrane. PS exposed on the inner membrane resides on the outer membrane of vesicles formed by degradation of the lipid bilayer. Consequently, they can immobilize Annexin V-APC molecules and fluoresce at 660 nm, which can be detected by flow cytometry.

[0279] During apoptosis, cells subsequently undergo necrosis. The nuclear envelope degrades, and DNA is released into the culture medium. PI can intercalate between the bases of DNA and emit a secondary fluorescence at 370-550 nm, which can also be detected by flow cytometry.

[0280] Briefly, after incubating cells with Ara-N3 probe at different concentrations and for different times (24 hours and 48 hours), the cells were recovered, trypsinized (for adherent cells) and washed in PBS. After counting with trypan blue on a Malassez cell, the cells were incubated in labeling buffer (100 μL) at room temperature in the dark for 15 minutes in the presence of annexin V-APC (5 μL) and IP (10 μL). After adding 100 μL of labeling buffer, the cells were directly analyzed by cytometer.

[0281] Table 5 below summarizes the non-toxic concentrations of Ara-N3 relative to the test cells.

[0282] B-In vivo experiments

[0283] Materials and methods

[0284] The Ara-N3 probe was prepared as detailed above.

[0285] Ten female NMRI nude mice (6 weeks old, Janvier, Le Genest-Saint Isle, France) were enrolled in the assay. Three mice were intravenously injected with 50 mg / mouse / injection of Ara-N3 every other day for 8 days (4 injections), for a cumulative dose of 200 mg. Another three mice were treated with 50 mg of Ara-N3 in 8 mL of water daily for 8 days, for a cumulative dose of 400 mg. As a control group, four tumor-bearing mice were not treated.

[0286] At the end of treatment, mice were euthanized and autopsied. Heart, lungs, brain, uterus, ovaries, kidneys, skeletal muscle, spleen, pancreas, abdominal fat, liver, skin, stomach, colon, cecum, small intestine, mesenteric lymph nodes, axillary lymph nodes, brachial lymph nodes, and whole blood were obtained and frozen for analysis.

[0287] result

[0288] Introducing Ara-N3 (up to 6.25 mg / mL) in the drinking water did not change the water intake of the mice.

[0289] Treatment with Ara-N3, either in the drinking water or via intravenous injection, did not cause any weight loss.

[0290] Table 5 (in vitro / in vivo)

[0291]

[0292] Example 6 :Specific labeling of tumor cell lines (in vivo) by Ara-N3 probe

[0293] Materials and methods

[0294] All animal experiments were performed in accordance with the European Economic Community's institutional guidelines for the use of laboratory animals (EU Directive 2010 / 63 / EU) and were authorized by the French Ministry of Higher Education and Research, reference APAFIS#8854-2017031314338357.

[0295] Subcutaneous tumor xenografts

[0296] Female NMRI nude mice (6 weeks old, Janvier, Le Genest-Saint Isle, France) were injected subcutaneously in the flank with 8×10 6 Panc-1 cells.

[0297] Ara-N3 treatment by intravenous injection: A cumulative dose of 20 mg Ara-N3 was evaluated.

[0298] Ten days after tumor cell implantation, Panc-1 tumor-bearing mice were treated with 1.8 mg / mouse / injection of Ara-N3 every other day for 21 days (11 injections), with a cumulative dose of 20 mg (n=3 mice per dose).

[0299] Untreated: As a control group, three tumor-bearing mice of each cell line were left untreated (Ctr).

[0300] Contrast agent administration

[0301] DBCO-IRDye800CW

[0302] Mice were treated with intravenous injection

[0303] - 24 hours after the last treatment injection (Ara-N3+Fluo), two Panc-1 tumor-bearing mice per treatment condition were injected intravenously with 100 μg of DBCO-IRDye800CW.

[0304] Control mice

[0305] -An untreated Panc-1 tumor-bearing mouse was injected intravenously with 100 μg of DBCO-IRDye800CW (Ctr+Fluo).

[0306] Fluorescence imaging data analysis (ex vivo)

[0307] Mice were euthanized and dissected 48 hours after DBCO-IRDye800CW injection, and ex vivo fluorescence imaging of skeletal muscle and tumors was performed.

[0308] Semiquantitative data were obtained from fluorescence images by manually drawing a region of interest (ROI) adjusted over the tumor area to be quantified. The total fluorescence signal measured in the ROI was divided by the number of pixels in the ROI. Therefore, data were expressed as relative fluorescence units (RLU) per pixel for both skeletal muscle and tumor. The ratio of fluorescence signals between tumor and skeletal muscle (tumor / muscle ratio) was then calculated.

[0309] The results are as follows Figure 1 shown.

Claims

1. An in vitro method for labeling or targeting tumor eukaryotic cells from a multicellular organism, wherein the tumor eukaryotic cells are selected from bladder cancer cells, blood cancer cells, colon cancer cells, gastric cancer cells, breast cancer cells, lung cancer cells, skin cancer cells and pancreatic cancer cells, the method comprising the following steps: a) contacting a sample comprising eukaryotic cells with at least one modified monosaccharide compound; b) contacting the sample of step (a) with a compound bearing a first reactive group, optionally in the presence of copper; as well as c) optionally detecting the binding of the compound of step (b) to the monosaccharide of step (a) to detect eukaryotic cells; wherein the at least one modified monosaccharide compound has the following formula (I): wherein X is a second reactive group, the first reactive group and the second reactive group are capable of reacting together in a click chemistry reaction, wherein the method is not a diagnostic method or a therapeutic method, and wherein the at least one modified monosaccharide compound of formula (I) is 5-azido-5-deoxy-D-arabinofuranosyl having the formula:

2. A pharmaceutical composition comprising a eukaryotic cell having at least one modified monosaccharide compound of formula (I) present on its surface, the modified monosaccharide compound of formula (I) optionally being operably linked to an anticancer drug or a particle comprising an anticancer drug, wherein the eukaryotic cell is an isolated non-cancerous eukaryotic cell and the at least one modified monosaccharide compound of formula (I) is 5-azido-5-deoxy-D-arabinofuranosyl having the formula:

3. The composition of claim 2, wherein the anticancer drug is selected from the group consisting of chemotherapeutic drugs, anticancer antibodies and kinase inhibitors.

4. The composition according to claim 2 or 3, wherein the cells are pluripotent stem cells or T cells.

5. Use of the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating cancer, wherein the cancer is selected from bladder cancer, blood cancer, colon cancer, stomach cancer, breast cancer, lung cancer, skin cancer and pancreatic cancer.

6. Use of the pharmaceutical composition according to claim 2 in the preparation of a medicament for diagnosing cancer, wherein the cancer is selected from bladder cancer, blood cancer, colon cancer, stomach cancer, breast cancer, lung cancer, skin cancer and pancreatic cancer.

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