EP4200613A1 - Composé monosaccharidique pour le marquage de la sécrétion cellulaire - Google Patents

Composé monosaccharidique pour le marquage de la sécrétion cellulaire

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Publication number
EP4200613A1
EP4200613A1 EP21763340.3A EP21763340A EP4200613A1 EP 4200613 A1 EP4200613 A1 EP 4200613A1 EP 21763340 A EP21763340 A EP 21763340A EP 4200613 A1 EP4200613 A1 EP 4200613A1
Authority
EP
European Patent Office
Prior art keywords
cell secretion
compound
labelling
reactive group
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21763340.3A
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German (de)
English (en)
Inventor
Sam Dukan
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Theraonco
Original Assignee
Theraonco
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Publication date
Application filed by Theraonco filed Critical Theraonco
Publication of EP4200613A1 publication Critical patent/EP4200613A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the present invention relates to the medicinal field, in particular of oncology. It relates to monosaccharide compounds for use in the labelling and/or diagnosis of cell secretion.
  • Carbohydrates are important as signalling molecules and for cellular recognition events. They can produce multivalent interactions with carbohydrate recognition proteins (CRPs) and be used as probes of living organisms. Carbohydrates thus present many opportunities in disease diagnosis. As a consequence, the development of carbohydrate-based bioactive compounds and sensors has become an active research area. An effective and modular synthetic approach to prepare functional carbohydrates derivatives is click chemistry. In that respect, WO2016/177712 describes modified monosaccharide compounds, such as 5-azido- 5-deoxy-D-arabinofuranose (also called herein “Arabinose-Ns” or “Ara-Ns”) in methods for labelling specifically living microorganisms.
  • 5-azido- 5-deoxy-D-arabinofuranose also called herein “Arabinose-Ns” or “Ara-Ns”
  • modified carbohydrate/monosaccharide compounds used as probes have never been described as being themselves diagnostic agents directed to cells secretion and dissemination.
  • Cell secretions are associated to various diseases such as cancer and neurodegenerative diseases. Their potential as a source of biomarkers, such as diagnostic biomarkers, is an area of intense interest. There is therefore a need to find and develop new candidates for the labelling and/or concentrating and/or targeting and/or diagnosis of cell secretions.
  • modified monosaccharide compounds that can specifically label cell secretion, which can be useful in diagnosis of various diseases, such as cancer.
  • the present invention is based on a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is (i) a reactive group adapted for click chemistry to a labelling agent or (ii) a labelling agent, for use in the labelling and/or concentrating and/or targeting and/or diagnosis of cell secretion.
  • R is (i) a reactive group adapted for click chemistry to a labelling agent or (ii) a labelling agent, for use in the labelling and/or concentrating and/or targeting and/or diagnosis of cell secretion.
  • the present invention also relates to a pharmaceutical composition comprising at least one compound of formula (I) or a metabolite thereof, in a pharmaceutically acceptable support for use in the labelling or diagnosis of cell secretion. It also relates to a method for labelling or concentrating or detecting cell secretion, the method comprising the steps of: (a) contacting a biological sample with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is (i) a reactive group adapted for click chemistry to a labelling agent or (ii) a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting cell secretion by labelling.
  • FIG. 1 - Lung tissue Cell secretion labelling between the primary lung cells (B- fluorescence imaging- and D-phase contrast) and the A549 cell line (A-fluorescence imaging- and C-phase contrast) after labelling by the Ara-N 3 (or +XN3) probe. Cell secretion labelling of the primary lung cells (E-fluorescence imaging- and F-phase contrast) without Ara-N3 (or - XN3) probe.
  • Figure 2 Labelling of AGS cells secretion by the Ara-N 3 (or +XN3) probe with DMEM growth medium (A-fluorescence imaging- and C-phase contrast) and with primary cell medium (B-fluorescence imaging- and D-phase contrast). Labelling of AGS cells secretion without the Ara-N 3 (or -XN3) probe with DMEM growth medium (E-fluorescence imaging- and G-phase contrast) and with primary cell medium (F-fluorescence imaging- and H-phase contrast).
  • Figure 3 Labelling of HT29 cells secretion by the Ara-N3 (or +XN3) probe with DMEM growth medium (A-fluorescence imaging- and C-phase contrast) and with primary cell medium (B-fluorescence imaging- and D-phase contrast). Labelling of HT29 cells secretion without the Ara-N3 (or -XN3) probe with DMEM growth medium (E-fluorescence imaging- and G-phase contrast) and with primary cell medium (F-fluorescence imaging- and H-phase contrast).
  • Figure 4 Labelling of A549 cells secretion by the Ara-N3 (or +XN3) probe with DMEM growth medium (A-fluorescence imaging- and C-phase contrast) and with primary cell medium (B-fluorescence imaging- and D-phase contrast). Labelling of A549 cells secretion without the Ara-N3 (or -XN3) probe with DMEM growth medium (E-fluorescence imaging- and G-phase contrast) and with primary cell medium (F-fluorescence imaging- and H-phase contrast).
  • subject encompasses non- human animals such as domestic or farm animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others.
  • diagnosis refers to the determination as to whether a subject is likely to be affected by a disease, preferably such as cancer.
  • diagnosis markers the presence, absence, or amount of which is indicative of the presence or absence of a disease.
  • diagnosis it is also intended to refer to the provision of information useful for the diagnosis of a disease, in particular such as cancer.
  • the terms “disorder” or “disease” refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infections, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors.
  • these terms refer to a health disorder or disease e.g. an illness that disrupts normal physical or mental functions.
  • the disease is a disease in which cell secretion is abnormal (i.e. increased or enhanced or decreased), preferably in comparison to a reference condition (e.g. healthy or normal conditions).
  • biological sample refers to any sample containing cells derived from a subject.
  • samples may be obtained from fluid sample such as blood, plasma, urine and seminal fluid samples as well as from biopsies, organs, tissues or cell samples.
  • cancer or “tumour”, as used herein, refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases). It can be a solid cancer or a hematopoietic cancer. Particular types of cancer are provided herein under the paragraph “Subject, regimen and administration”. “Cancer cells” or “tumour cells” are cells that divide relentlessly, forming solid tumours or flooding the blood with abnormal cells.
  • Cancer cells or cells suspected of being cancerous are preferably cells that display one or more cancer cell characteristics, e.g., immortalization, loss of contact inhibitions, reduced cellular adhesion, invasiveness, loss of anchorage dependence, lower serum requirements, molecular changes in cell membrane components, disorganization of the cytoskeleton, and/or defective electrical communication.
  • cancer sample or “tumour sample” refers to any sample containing tumour cells and/or tumour cell secretion derived from a subject.
  • tumour samples may be obtained from fluid sample such as blood, plasma, urine and seminal fluid samples as well as from biopsies, organs, tissues or cell samples.
  • anti-cancer therapy As used herein, the terms “anti-cancer therapy”, “anti-cancer treatment” and “anticancer agents” are used interchangeably and refer to compounds which are used in the treatment of cancer, in particular surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy or any combinations thereof.
  • cell secretion or “cell secretion products” refer to secretory vesicles which transiently dock and fuse to release intra-vesicular contents from cells. Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell.
  • cell secretion products are said membranous sacs or vesicles that can be extracellular vesicles, oncosomes, exosomes, ectosomes and/or mucus.
  • kit defines especially a "kit of parts” in the sense that the combination partners (a) and (b), as defined in the present application can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e. simultaneously or at different time points. The parts of the kit of parts can then be administered simultaneously or chronologically staggered, that is at different time points for any part of the kit of parts.
  • the ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can vary.
  • the combination partners (a) and (b) can be administered by the same route or by different routes.
  • the reactive group R of the compound of formula (I) is a reactive group generally used in click chemistry. Click chemistry is a well-known method from a skilled person for attaching a probe or a substrate of interest to a specific biomolecule, such as a modified monosaccharide compound. Click chemistry generally implements biorthogonal reactions.
  • the reactive group R can be defined as a reactive group involved in biorthogonal reactions.
  • a number of chemical ligation strategies have been developed that fulfil the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry), between nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, the tetrazine ligation, the isocyanide- based click reaction, and most recently, the quadricyclane ligation.
  • an azide alkyne cycloaddition is a well-known so-called click chemistry reaction, in the presence or not of a copper catalyst, in which the azide group reacts with the alkyne group to afford a triazole.
  • the alkyne group (-C ⁇ C-) can be strained or not.
  • the alkyne group can be a terminal alkyne (i.e.:-C ⁇ CR’, where R’ is H or a (C1-C6) alkyl group, the alkyl group being for instance methyl, ethyl, propyl, isopropyl) or the alkyne group can be a strained alkyne, and more specifically a cyclic strained alkyne, such as cyclooctynes.
  • the present invention includes within its scope all stereoisomeric and isomeric forms of the compounds disclosed herein, including all diastereomeric isomers, racemates, enantiomers and mixtures thereof.
  • E and Z isomers also known as cis and trans isomers.
  • the present disclosure should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+), and/or (-) forms of the compounds, as appropriate in each case.
  • a structure has no specific stereoisomerism indicated, it should be understood that any and all possible isomers are encompassed.
  • Compounds of the present invention embrace all conformational isomers.
  • Compounds of the present invention may also exist in one or more tautomeric forms, including both single tautomers and mixtures of tautomers.
  • the term “comprise(s)” or “comprising” is “open-ended” and can be generally interpreted such that all of the specifically mentioned features and any optional, additional and unspecified features are included. According to specific embodiments, it can also be interpreted as the phrase “consisting essentially of” where the specified features and any optional, additional and unspecified features that do not materially affect the basic and novel characteristic(s) of the claimed invention are included or the phrase “consisting of” where only the specified features are included, unless otherwise stated.
  • the term “and/or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.
  • the term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • the compound of formula (I) includes any diastereoisomer thereof.
  • the compound of formula (I) is selected in the group consisting of the following formulae: wherein R is (i) a reactive group adapted for click chemistry with a labelling agent or (ii) a labelling agent.
  • R can be for instance an azide (-N3) or an alkyne group.
  • R is a reactive group suitable for click chemistry, as detailed above.
  • R is more specifically selected among groups consisting in or bearing the azido group (-N3) and groups consisting in or bearing the alkyne group (-C ⁇ C-). R is thus more specifically a group usually involved in an azide alkyne cycloaddition.
  • Click chemistry is a well-known method from a skilled person for attaching a probe or a substrate of interest to a specific biomolecule, such as a modified monosaccharide compound according to the invention.
  • An azide alkyne cycloaddition is a well-known so-called click chemistry reaction, in the presence or not of a copper catalyst, in which the azide group reacts with the alkyne group to afford a triazole.
  • the alkyne group can be strained or not.
  • Such azide alkyne cycloaddition can be performed in copper catalyzed conditions in the presence of a ligand, preferably a tris-triazole ligand such as TGTA (Tris((l-( -D- glucopyranosyl)-l [l,2,3]-triazol-4- yl)methyl)amine) or TBTA (Tris-[(l-benzyl-l l,2,3- triazol-4-yl) methyl]amine).
  • TGTA Tris((l-( -D- glucopyranosyl)-l [l,2,3]-triazol-4- yl)methyl)amine
  • TBTA Tris-[(l-benzyl-l l,2,3- triazol-4-yl) methyl]amine
  • THPTA tris(3- hydroxypropyl triazolylmethyl)amine
  • BTTES 2-(4-((bis((l-tert-butyl-l 1,2,3-triazol- 4- yl)methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)ethanesulfonic acid
  • BTTES tris((l- ((( ethyl) carboxymethyl)-(l,2,3-triazol-4-yl)) methyl) amine, bathophenanthroline disulfonate, or tris(2-benzimidazolylmethyl)amines.
  • azide alkyne cycloaddition can be performed in the absence of copper, if a strained alkyne is used, such as azadibenzocyclooctyne (ADIBO, DIBAC or DBCO) or tetramethoxydibenzocyclooctyne (TMDIBO).
  • ADIBO azadibenzocyclooctyne
  • DIBAC DIBAC
  • DBCO tetramethoxydibenzocyclooctyne
  • strained alkynes frequently used for copper-free reaction include: cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa- dibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne.
  • OCT cyclooctyne
  • ALO aryl-less cyclooctyn
  • R is a strained alkyne, such as azadibenzocyclooctyne (ADIBO, DIBAC or DBCO) or tetramethoxydibenzocyclooctyne (TMDIBO).
  • ADIBO azadibenzocyclooctyne
  • DIBAC DIBAC
  • DBCO azadibenzocyclooctyne
  • TMDIBO tetramethoxydibenzocyclooctyne
  • strained alkynes frequently used for copper-free reaction include: cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa- dibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne.
  • OCT cyclooctyne
  • ALO aryl-less cyclooctyn
  • R can thus be any one of the first or second reactive groups as specified above, the other group, defined below as X, is thus one of the second or first reactive groups, respectively.
  • R is an alkyne group of formula: -C ⁇ CR’, where R’ is H or a (C1-C6)alkyl group, the alkyl group being linear, cyclic or branched, including, but not limited to, methyl, ethyl, propyl, isopropyl.
  • R’ is H.
  • R is a strained alkyne, and more specifically a cyclic strained alkyne, such as cyclooctynes.
  • R can be selected in the group consisting of azadibenzocyclooctyne (ADIBO, DIBAC or DBCO) or tetramethoxydibenzocyclooctyne (TMDIBO).
  • Other appropriate strained alkynes frequently used for copper-free reaction include: cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa-dibenzocycloocty
  • R is selected among groups consisting in or bearing the azido group (-N 3 ). More specifically, R is the azido group.
  • the compound of formula (I) is selected in the group consisting of the following formulae:
  • the compound of formula (I) is 5-azido-5-deoxy-D- arabinofuranose (arabinose-N 3 or also named Ara-N 3 ), in particular having the following formula (II):
  • the compound of formula (I) has the following formula (III): wherein CCR’ is an alkyne group as defined above, and more specifically R’ is H or a (C1-C6)alkyl group, the alkyl group being linear, cyclic or branched, including, but not limited to, methyl, ethyl, propyl, isopropyl.
  • R’ is H.
  • the compound for use according to the invention is a metabolite of a compound of formula (I), more specifically a metabolite of a compound of formula (II), or (III).
  • the metabolite is a metabolite of arabinose, preferably a metabolite of L-arabinose.
  • a “metabolite of a compound of formula (I)” is a compound from the pentose phosphate pathway, wherein said compound further comprises a reactive group R, as defined above, and more specifically R is selected among groups consisting in or bearing the azido group (-N3) and groups consisting in or bearing the alkyne group (-C ⁇ C-).
  • R is selected among groups consisting in or bearing the azido group (-N3) and groups consisting in or bearing the alkyne group (-C ⁇ C-).
  • a metabolite of a compound of formula (I) includes ribose, D-ribose, L-ribose, ribose 5-P, D-ribose 5-P, D- ribose 1-P, D-ribose 1,5-P, ribulose, ribulose 5-P, L-ribulose, L-ribulose 5-P, D-ribulose, D- ribulose 5-P, arabinitol, L-arabinitol, xylulose, xylulose-5-P, D-xylulose, D-xylulose-5-P, L- xylulose, xylose, D-xylose, L-xylose, and xylitol, said compound further comprising an azide (N 3 ) or an alkyne group, as defined above.
  • a metabolite of a compound of formula (I) is a metabolite of arabinose, further comprising an azide (N3) or an alkyne group, as defined above.
  • the compounds of the pentose phosphate pathway used in the present invention are selected in the group consisting of ribose, D-ribose, L-ribose, ribose 5-P, D-ribose 5-P, D-ribose 1-P, D-ribose 1,5-P, ribulose, ribulose 5-P, L-ribulose, L-ribulose 5-P, D-ribulose, and D-ribulose 5-P, said compound further comprising an azide (N 3 ) or an alkyne group, as defined above.
  • the compounds of the pentose phosphate pathway used in the present invention are selected in the group consisting of ribose, D-ribose, and L-ribose, said compound further comprising an azide (N3) or an alkyne group, as defined above.
  • the compounds disclosed herein comprise or are detectable moiety, namely moiety consisting in or bearing a detectable substance (or a labelling agent), namely a substance capable to be detected by techniques known by one skilled in the art, such as fluorescence, colorimetry, luminescence or autoradiography.
  • the imaging techniques can thus be fluorescence, magnetic resonance or computed tomography.
  • R is either (i) a reactive group adapted for click chemistry to obtain a compound with a labelling agent or (ii) a labelling agent.
  • the compound according to the invention allows a direct or indirect labelling of cell secretion.
  • detectable substances or labelling agent include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35 S, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors); luminescent labels (e.g.
  • the labelling agent is a dye.
  • the dyes can be selected from the group consisting of fluorescent, luminescent or phosphorescent dyes, preferably dansyl, fluorescein, acridine, rhodamine, coumarin, BODIPY and cyanine dyes. More specifically, the fluorescent dyes can be selected among the dyes marketed by Thermo Fisher such as the Alexa Fluor dyes, Pacific dyes or Texas Red or by other providers for cyanines 3, 5 and 7. In particular, dyes bearing azide for CuAAC are commercially available for Alexa Fluor® 488, 55, 594 and 647 and for TAMRA (tetramethylrhodamine). In a second aspect, the labelling agent is a radiolabel.
  • the radioactive label provides for an adequate signal and has sufficient half-life.
  • the labelling agent is a radioisotope.
  • radioisotopes that may be used in the present invention are the following: 277 Ac, 211 At, 128 Ba, 131 Ba, 7 Be, 204 Bi, 205 Bi, 206 Bi, 76 Br, 77 Br, 82 Br, 109 Cd, 47 Ca, 11 C, 14 C, 36 Cl, 48 Cr, 51 Cr, 62 Cu, 64 Cu, 67 Cu, 165 Dy, 155 Eu, 18 F, 153 Gd, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 198 Au, 3 H, 166 Ho, 111 In, 113m In, 115m In, 123 I, 125 I, 131 I, 189 Ir, 191m Ir, 192 Ir, 194 Ir, 52 Fe, 55 Fe, 59 Fe, 177 Lu, 15 O, 119m-191 Os, 109 Pd, 32 P, 33 P, 42
  • the radioisotope is imaged with a photoscanning device.
  • 99m Tc-labeling procedures are particularly described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982).
  • the detectable substance or moiety can be an affinity tag.
  • Such an affinity tag can be for instance selected from the group consisting of biotin, His-tag, Flag-tag, strep- tag, sugars, lipids, sterols, PEG-linkers, and co-factors.
  • the detectable substance is a biotinylated label.
  • Biotins linked to azide are commercially available (Biotin azide). More particularly, biotin can be detected by an antibody or another protein specific to said first ligand (such as avidin), said antibody or protein bearing a detectable substance or moiety, preferably a fluorochrome or luminescent molecule or an enzyme or a radiolabel.
  • the detectable substance is biotin, so that in the compound of formula I, R is biotin.
  • a labelled antibody for example a fluorescent antibody, directed to or against biotin may be used to reveal the presence of the compound according to the invention.
  • anti-biotin antibody may particularly be labelled with Alexa Fluorochrome 488.
  • the labelling agent is a fluorescent label or a radiolabel.
  • R is a labelling agent (or a label), preferably a detectable substance or moiety such as described above.
  • the compound of formula I or its metabolite bears a reactive group R which is complementary with a second reactive group X, both reactive groups X and R reacting together in a click chemistry reaction, preferably an azide alkyne cycloaddition.
  • the compound bearing the second reactive group X comprises or is a directly detectable moiety or comprises or is an indirectly detectable moiety.
  • the compound bearing the second reactive group X can be included in or be part of magnetic beads.
  • the chemical reaction results in a covalent link.
  • R is an azido group (-N 3 ) or a group bearing an azido group
  • the second reactive group X is an alkyne or a group bearing an alkyne group.
  • the second reactive group is an azido group (-N 3 ) or a group bearing an azido group.
  • the second reactive group X is an azido group (- N3) and the first reactive group is an alkyne group (-C ⁇ C-).
  • compositions comprising at least one compound of formula (I) or a metabolite thereof as above defined, in a pharmaceutically acceptable support, for use in the labelling and/or targeting and/or concentrating and/or diagnosis of cell secretion.
  • the compound of the invention as defined above is a diagnostic active agent, or preferably the sole diagnostic active agent.
  • the compound of the invention as defined above is a direct or indirect labelling agent, which can be useful for the diagnosis of diseases, such as cancers.
  • the compositions contemplated herein include a pharmaceutically acceptable carrier with at least one compound of formula (I).
  • pharmaceutically acceptable carrier is meant to encompass any carrier (e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host or sample to which it is administered or contacted.
  • the pharmaceutical compositions can be formulated to be suitable for the route of administration. Particularly, the pharmaceutical composition is formulated to be administered enterally or parenterally.
  • the pharmaceutical composition can be orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, or regionally administered. Administration includes direct injection or perfusion.
  • the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
  • the pharmaceutical composition is a sterile injectable composition.
  • a sterile injectable composition e.g., a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) or suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • compositions suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient.
  • a composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • the carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
  • the pharmaceutical compositions are advantageously applied by injection or intravenous infusion of suitable sterile solutions. Methods for the safe and effective administration of most of diagnosis compositions are known to those skilled in the art. In addition, their administration is described in the standard literature. Subject and administration The compound or pharmaceutical composition disclosed herein can be administered to a subject. Preferably, the compound or composition are administered in effective amounts.
  • an “effective amount” is that amount of the labelling agent conjugated to a compound of formula I that alone, or together with further doses/compounds, signals presence of cell secretion. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed below. Effective amounts will depend, of course, on the subject parameters including age, physical condition, size, gender and weight, the duration of the administration, the specific route of administration and like factors within the knowledge and expertise of the man skilled in the art or the health practitioner. It is interesting to note that the monosaccharide compound of formula (I) or metabolite thereof present a low or no toxicity towards cells, so that the used amount thereof can vary in a large range.
  • the concentration of the monosaccharide compound of formula (I) or its metabolite of the invention can vary from 10 ⁇ M to 100mM, preferably from 1mM to 50mM, more preferably from 1mM to 20mM.
  • the compound or the composition described herein can be administered orally, parenterally, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or via inhalation spray.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • the compound or composition of the invention may be administered as a single dose or in multiple doses.
  • the compound or composition of the invention is administered prior to the targeting and/or concentrating and/or diagnosis method, preferably within 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days, prior to targeting and/or concentrating and/or diagnosis (e.g. imaging, detection and/or measurement of labelling).
  • the compound of formula I or its metabolite can be administered by a first route and the compound bearing the second reactive group X adapted for click chemistry can be administered by the same or another route of administration.
  • the compound of formula I or its metabolite and the compound bearing the second reactive group X can be administered sequentially, separately or simultaneously.
  • the compound of formula I or its metabolite and the compound bearing the second reactive group X are both administered to the patient, i.e. used in vivo.
  • sequential means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of the monosaccharide compound disclosed herein and the compound bearing the second reactive group X, a sequential dosage regimen could include administration of the monosaccharide compound of the invention before, simultaneously, substantially simultaneously, or after administration of the compound bearing the second reactive group X, but both agents will be administered in a regular sequence or order.
  • sequential dosage regimen could include administration of the monosaccharide compound of the invention before, simultaneously, substantially simultaneously, or after administration of the compound bearing the second reactive group X, but both agents will be administered in a regular sequence or order.
  • the term “separate” means, unless otherwise specified, to keep apart one from the other.
  • the term “simultaneously” means, unless otherwise specified, happening or done at the same time, i.e., the monosaccharide compound of the invention and the compound bearing the second reactive group X are administered at the same time.
  • substantially simultaneously means that the monosaccharide compound of the invention and the compound bearing the second reactive group X are administered within minutes of each other (e.g., within 15 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two compounds separately).
  • R is a reactive group for click chemistry
  • the compound of formula I or its metabolite is administered to the patient by any suitable route.
  • a biological sample from said patient is provided, for example such as a blood or urine sample.
  • the compound bearing a reactive group able to react with the reactive group R by click chemistry is then contacted ex vivo or in vitro with the biological sample to label cell secretion.
  • only the monosaccharide according to the invention is administered to the patient (i.e. used in vivo) and then the compound bearing a reactive group able to react with the reactive group R by click chemistry is applied to the biological sample from the patient (i.e. used ex vivo or in vitro).
  • the cell secretion products are isolated, such as by filtration (e.g. with cell strainer), from the biological sample obtained from the patient, and then the isolated cell secretion products are labelled or concentrated.
  • R is a labelling agent (or a label), preferably a detectable substance or moiety such as described above
  • the compound of formula I or its metabolite is administered to the patient by any suitable route.
  • a biological sample from the patient is provided, for example such as a blood or urine sample and can be analysed ex vivo or in vitro for further studies.
  • the cell secretion products are isolated, for instance by filtration (e.g.
  • the subject is a mammal, even more preferably a human.
  • the human subject according to the invention may be a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, preferably an adult of at least 40 years old, still more preferably an adult of at least 50 years old, even more preferably an adult of at least 60 years old.
  • the subject can be suffering from any disease in which cell secretion is altered (enhanced or decreased, for example such as cancer) or in which cell secretions are involved in the disease onset, progression, dissemination and outcome (for example such as cancer or neurodegenerative disease).
  • the disease is selected from the group consisting of cancer, neurodegenerative disease, autoimmune disease, infectious disease such as bacterial or viral infection and cardiovascular disease.
  • the subject has been diagnosed with a cancer or is suspected to have cancer.
  • a subject having cancer can be identified by routine medical procedures prior to the methods provide herein.
  • a subject suspected of having cancer may show one or more symptoms associated with certain types of cancers. Cancer symptoms vary, depending upon the types of cancers.
  • the cancers may comprise non solid tumours (such as haematological tumours, for example, leukaemia and lymphoma) or may comprise solid tumours.
  • solid tumour is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
  • Typical cancers are solid or hematopoietic cancers such as breast, brain, stomach, liver, skin, prostate, pancreatic, oesophageal, sarcoma, ovarian, endometrium, bladder, cervix uteri, rectum, colon, renal, lung or ORL cancers, paediatric tumours (neuroblastoma, glioblastoma multiforme), lymphoma, carcinoma, glioblastoma, hepatoblastoma, leukaemia, myeloma, seminoma, Hodgkin or malignant hemopathies.
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • the subject suffers or is suspected to suffer from liver cancer and cholangiocarcinoma, nasopharyngeal cancer, lung cancer, gastric cancer, spongioblastoma, prostate cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, melanoma, pancreatic cancer, renal cell carcinoma, stomach cancer.
  • the subject has already received at least one line of anticancer treatment, by any method known by the skilled person in the art, e.g. surgery, chemo- therapy, radiotherapy or combination thereof.
  • a compound or a composition such as described herein comprising or conjugated with a labelling agent can be used for detecting presence of cancer cell secretion.
  • a suitable amount of the compound or composition according to the invention can be injected to a suspected cancer patient, e.g., a patient carrying or suspected of carrying a tumour, by any suitable route, for example such as described herein.
  • the patient is then subjected to scintigraphy or fluorometry at suitable periods, e.g., 2 h, 4 h, 24 h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days after injection. Labelling (e.g.
  • radioactivity or fluorescence of the whole body and the regions of interest are preferably normalized against background activity, and the presence/absence of tumour matter can be determined based on the results thus obtained.
  • R is a reactive group for click chemistry
  • the compound of formula I or its metabolite is administered to the patient by any suitable route for detecting presence of cancer cell secretion.
  • a biological sample from the patient is provided, for example such as a blood or urine sample.
  • the compound bearing the second reactive group X is then contacted ex vivo or in vitro with the biological sample to label cancer cell secretion.
  • only the monosaccharide according to the invention is administered to the patient (i.e.
  • the subject has been diagnosed with a neurodegenerative disease or is suspected to have a neurodegenerative disease.
  • the neurodegenerative disease may be selected from the group consisting of Parkinson’s disease, Alzheimer’s disease, Down syndrome, Prion Diseases, Huntington's disease and Amyotrophic Lateral Sclerosis.
  • the subject has been diagnosed with an autoimmune disease or is suspected to have an autoimmune disease.
  • the autoimmune disease can be selected from the group consisting of Type 1 diabetes mellitus, Multiple sclerosis, Systemic lupus erythematosus, Rheumatoid arthritis and Sjogren’s syndrome.
  • the subject has been diagnosed with an infectious disease or is suspected to have an infectious disease.
  • the infectious disease is a bacterial infection such as tuberculosis, diphtheria, and toxoplasmosis or is an infection caused by prions or viruses such as HIV.
  • the subject has been diagnosed with a cardiovascular disease or is suspected to have a cardiovascular disease.
  • Cardiovascular disorders may include atherosclerosis, ischaemic (coronary) heart disease, myocardial ischaemia (angina), myocardial infarction, aneurismal disease, atheromatous peripheral vascular disease, aortoiliac disease, chronic and critical lower limb ischaemia, visceral ischaemia, renal artery disease, cerebrovascular disease, stroke, atherosclerotic retinopathy, thrombosis and aberrant blood clotting, and hypertension, including pulmonary arterial hypertension.
  • Method for labelling, detecting or targeting cell secretion As above detailed, the compound of formula (I) or metabolite thereof, is a label or is suitable for forming a detectable entity with a label, preferably a fluorescent or radioactive label.
  • the present invention enables labeling of cell secretion products, numbering or detecting cell secretion products as well as concentrating and/or isolating cell secretion products, optionally immobilized on a solid support; especially with a solid support constituted of magnetic beads bearing the second reactive group X as detailed above.
  • the method may be implemented with any sample, typically a biological sample of a subject, e.g. a fluid, such as a sample of blood, plasma, serum, urine, cerebrospinal fluid or a sample from a tissue of a subject or a part thereof.
  • the invention may be implemented with samples from any subject, including any human patient having or suspected to have cancer.
  • each sample used at step (a) of the method comprises a cell population or preferably an individual cell, preferably obtained by cell sorting, in particular by flow cytometry.
  • the method can be implemented to the whole body of the subject or a part thereof.
  • the monosaccharide compound of formula (I) or a precursor thereof and optionally the compound bearing a first reactive group can be administered enterally (including orally) or parenterally (including intravenously or intramuscularly).
  • An aspect of the present invention is a method, preferably an in vitro or ex vivo method, for labelling and/or concentrating or detecting or targeting cell secretion, the method comprising the steps of: (a) contacting a biological sample with a compound of formula (I) or metabolite thereof such as disclosed herein, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group able to react with the reactive group R by click chemistry, optionally in presence of copper; d) detecting cell secretion
  • the step a) according to the method for labelling, concentrating, detecting or targeting secretion of cells comprises contacting a biological sample comprising cells susceptible or able to produce cell secretion, with at least one compound of formula (I) or a metabolite thereof, such as disclosed herein.
  • Such contacting step a) allows the incorporation of the at least one compound of formula (I) or a metabolite thereof in a biological sample comprising cells susceptible or able to produce cell secretion products, such as extracellular vesicles, oncosomes, exosomes, ectosomes and/or mucus.
  • the biological sample can be a tissue, an organ, blood, plasma, saliva, urine, serum, breast milk, cerebrospinal and seminal fluid samples.
  • Such process may correspond to an assimilation of the compound of formula (I) or a metabolite thereof by the secreting cells.
  • Such secreting cells might then produce cell secretion products, such as extracellular vesicles, oncosomes, exosomes, ectosomes and/or mucus themselves, which, in turn, comprise the assimilated compound of formula (I) or a metabolite thereof.
  • the cell secretion products can be isolated from the biological sample of step (a) by any means, such as by filtration (e.g. with cell strainer), as to separate the cell secretion products from the secreting cells, for instance. Accordingly, the cell secretion products can be concentrated.
  • the method comprises a step (c) of contacting the sample of step (a) with a compound comprising a reactive group X adapted for click chemistry such as above defined.
  • step c) allows to generate the click chemistry reaction between the first reactive group R of the compound of formula I or its metabolite and the second reactive group X of the compound which is a direct or indirect labelling agent, thereby providing cell secretion that are labelled or targeted or can be labelled or targeted thereafter (as detailed above).
  • the compound of formula I or a metabolite thereof can be administered to a subject by any route of administration (i.e. in vivo).
  • step (a) comprises providing a biological sample from a subject which has been previously administered with a compound of formula (I) or metabolite thereof such as disclosed herein, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent. Then, the compound bearing a reactive group able to react with the reactive group R by click chemistry can be put in contact of cell secretion in vitro or ex vivo afterwards, as detailed above for optional steps (b) and (c), for example after providing a biological sample from the subject, and optionally after having isolated cell secretion from the biological sample.
  • the step c) is not necessary when R is a labelling agent itself.
  • steps (a), (b) or (c) One skill in the art knows how to implement steps (a), (b) or (c).
  • said steps (a) and/or (b) and/or (c) are carried out in culture, incubation or preservation media and/or conditions.
  • the culture media allows the growth and/ or survival of cells.
  • the cell culture medium can be supplemented by any compound to enhance or stimulate cells growth and/or survival and/or preservation and/or assimilation of the modified monosaccharide compound of formula (I).
  • the sample is a biological sample such as blood or urine, the sample is conserved under conditions (e.g. temperature, preservative compounds) suitable for its preservation.
  • step (a) allows the incorporation of the compound of formula (I) or a metabolite thereof in the secretion of cells and/or in the secreted cells.
  • steps (a) and/or (c) are carried out with reactants and/or catalysts for generating the reaction of the first reactive group R of the compound of formula (I) or metabolite thereof, with the compound comprising the second reactive group X.
  • the method may be implemented with any sample, typically a biological sample of a subject, e.g.
  • a fluid such as a sample of blood, plasma, serum, urine, cerebrospinal fluid or a sample from an organ or tissue of a subject or a part thereof having or suspected to have a disease, in particular a cancer, in particular such as disclosed below under the “Biological sample and reference sample” paragraph.
  • the method can be implemented to the whole body of the subject or a part thereof.
  • the compound of formula (I) or a metabolite thereof and optionally the compound bearing the second reactive group X can be administered by any suitable route, for example such as described above under the paragraph “Subject, regimen an administration”.
  • the method further includes a step d) comprising detecting the labelling agent or moiety.
  • detecting the labelling may include the visualizing, or detecting the presence of labelled cell secretion in a subject or in a biological sample, and/or the measuring of such labelling.
  • the measuring of such labelling such as fluorescence or radioactivity, allows to detect and/or evaluate cells secretion. Accordingly, the present invention enables labelling of cells secretion in a biological sample, numbering or detecting of cell secretion, as well as concentrating and/or isolating cell secretion, optionally immobilized on a solid support.
  • Detection and/or measure of the labelling can be performed by any methods known in the art.
  • fluorescence plate reader for example such as provided by Biotek, Synergy Mx, Colmar, France. Fluorescence quantification may be performed using fluorescence plate reader, for example such as provided by Biotek, Synergy Mx, Colmar, France.
  • the said detecting step d) can be carried out in a liquid medium or on a solid substrate.
  • the method of the present invention can further comprise one or more washing steps, preferably 2 washing steps, in particular prior to step d).
  • a cell secretion may be detected by radioautography or scintigraphy.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautography by various optical methods, or by counting the grains.
  • cell secretion can also be detected or “imaged” in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography.
  • a positron emission transaxial tomography scanner such as designated Pet VI located at Brookhaven National Laboratory, can also be used where the radiolabel emits positrons (e.g., 11 C, 18 F, 15 O, and 13 N).
  • the bio-distribution of the label can be monitored by scintigraphy. Whole body imaging techniques are particularly described in U.S. Pat. Nos.4,036,945 and 4,311,688.
  • time-resolved fluorometry can be used to detect a signal.
  • the method described in Christopoulos T K and Diamandis E P Anal Chem 1992:64:342-346 may be used with a conventional time-resolved fluorometer.
  • An aspect of the present invention is a method, preferably an in vitro or ex vivo method, for detecting, concentrating, selecting and/or recovering cell secretion, the method comprising the steps of: (a) contacting a biological sample with a compound of formula (I) or metabolite thereof such as disclosed herein, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group able to react with the reactive group R by click chemistry, optionally in presence of copper; d) detecting, selecting, concentrating or recovering cell secretion from step (a), (b) or (c).
  • step (a) the compound of formula I or a metabolite thereof can be administered to a subject by any route of administration (i.e. in vivo).
  • step (a) comprises providing a biological sample from a subject which has been previously administered with a compound of formula (I) or metabolite thereof such as disclosed herein, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent.
  • the compound bearing a reactive group able to react with the reactive group R by click chemistry can be put in contact of cell secretion in vitro or ex vivo afterwards, as detailed above for optional steps (b) and (c), for example after providing a biological sample from the subject, and optionally after having isolated cell secretion from the biological sample.
  • Such method can further comprise the analysis of cell secretion, in particular of the cell secretion content.
  • cell secretion content it is intended the composition, genomic or proteomic profile of the cell secretion.
  • Cell secretion comprises a wide variety of DNA, RNA, mRNA, miRNA, proteins lipids, metabolites, hormones, cytokines, even organelles or membrane. Such content may serve as biomarkers for a disease.
  • Biological sample and reference sample Method for diagnosing a disease
  • Any of the monosaccharide compound of formula (I) or metabolite thereof described herein can be used for the detection of cell secretion, in particular abnormal cell secretion (i.e. increased or decreased cell secretion in comparison to a reference sample condition), preferably cell secretion that can be correlated to a disease.
  • the disease can be any disease in which cell secretion is altered (enhanced or decreased, for example such as cancer) or in which cell secretions are involved in the disease onset, progression, dissemination and outcome (for example such as cancer or neurodegenerative disease).
  • the disease is selected from the group consisting of cancer, neurodegenerative disease, autoimmune disease, infectious disease such as bacterial or viral infection and cardiovascular disease.
  • diseases are particularly described above under the paragraph “Subject and administration”.
  • any of the monosaccharide compound of formula (I) or metabolite thereof described herein can be used for target delivery of a detectable label such as an imaging or labelling agent to cancer secretion cells, thereby facilitating cancer diagnosis.
  • the present invention then also relates to the use of a monosaccharide compound of formula (I) or a metabolite thereof as disclosed herein as a diagnosis tool for detecting cell secretion in a biological sample of a subject, the subject suffering from a disease or suspected to have a disease, in particular such as cancer.
  • the invention further relates to a monosaccharide compound of formula (I) or a metabolite thereof as disclosed herein, for medical imaging of cell secretion, preferably for diagnosis a cancer ultimately.
  • R is a labelling agent
  • the compound of formula (I) or a metabolite thereof is incubated with a sample producing or suspected of producing abnormal cell secretion.
  • Such a sample can be a sample containing cultured cells, a tissue sample obtained from a subject who has or is suspected of having a disease, or an in vivo tissue sample in such a subject.
  • R is a reactive group for click chemistry
  • the compound of formula (I) or a metabolite thereof and the compound bearing the second reactive group X can be administered to a subject who has or is suspected of having a disease, by any method or route of administration disclosed herein, in particular under the paragraph “Subject, regimen and administration”.
  • R is a reactive group for click chemistry
  • the compound of formula (I) or a metabolite thereof can be administered to a subject who has or is suspected of having a disease, by any method or route of administration disclosed herein, in particular under the paragraph “Subject, regimen and administration”, and the compound bearing a reactive group able to react with the reactive group R by click chemistry is subsequently applied on a biological sample provided from said treated patient, i.e. in vitro or ex vivo.
  • the diagnosis of the disease can either be implemented in vivo, ex vivo or in vitro.
  • the present invention further relates to a method, preferably an in vitro or ex vivo method, for identifying abnormal cell secretion, preferably as to diagnose a disease in a subject, comprising implementing a method for labelling or detecting or concentrating cell secretion as defined here above.
  • the method for diagnosis a disease in a subject comprises the step of detecting the labelling of cell secretion and comparing the labelling to a reference level.
  • the invention concerns a method, preferably an in vitro or ex vivo method, for identifying cell secretion, preferably as to diagnose a disease in a subject, comprising implementing a method for labelling or detecting or concentrating cancer cell secretion and/or of selecting or recovering cell secretion as defined here above.
  • the method for diagnosis a disease in a subject comprises the step of (i) detecting the labelling of cancer cell secretion and comparing the labelling to a reference level or (ii) analysing and comparing the cell secretion, preferably the cell secretion content, to a biological sample of reference.
  • the invention concerns a method, preferably an in vitro or ex vivo method, for identifying cancer cell secretion, preferably as to diagnose a cancer in a subject, comprising implementing a method for labelling or detecting or concentrating cancer cell secretion as defined here above.
  • the method for diagnosis a cancer in a subject comprises the step of detecting the labelling of cancer cell secretion and comparing the labelling to a reference level.
  • Detection and/or measure of the label is preferably performed from 10 hours to 40 hours, more preferably from 16 to 24, 25, or to 36 hours after implementing said method for labelling, and more specifically after implementing step (a) as detailed above.
  • higher measuring or “higher labelling” it denotes that the labelling ratio of the sample from a subject having or suspecting to have a disease with respect to a reference sample is more than 1.1, 1.2, 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.
  • the labelling ratio of the biological sample with respect to the reference sample is more 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.
  • the disease is diagnosed when the measuring of the labelled cell secretion is higher than a reference level.
  • the disease is diagnosed when the cell secretion, in particular the cell secretion content is different than the cell secretion, in particular the cell secretion content of a biological sample of reference.
  • the cell secretion may comprise a biomarker of a disease.
  • the content of the cell are particularly analysed, such as provide the genomic and/or proteomic profile of the cell secretion.
  • Jaworski et al. (Journal of Biological Chemistry (2014): jbc-M114) employed the RayBio Cytokine Array 1 to compare the cytokine profiles of exosomes released from T-cell lines that were infected with the human T-lymphotrophic virus type 1 (HTLV-1) with those from T-cells that were uninfected. They observed that exosomes from different cell lines had distinct cytokine profiles, with exosomes from HTLV-1 infected cells having elevated levels of GM-CSF and IL-6 and reduced levels of MCP-1 and RANTES. A number of exosomal protein biomarkers have been found to be potentially useful in the diagnosis of central nervous system diseases.
  • Such method may further comprises the use or the labelling of a biomarker of the disease, in particular to detect its presence in the cell secretion.
  • the method of diagnosing may comprise the detection of tau protein in the cell secretion.
  • the biological sample from a subject having or suspected to have a disease, particularly such as cancer, is as defined below, in particular under the paragraph “Biological sample and reference sample”.
  • the sample is more specifically suspected to comprise cancer cells secretion, in particular such as mucus, extracellular vesicles, oncosomes, ectosomes and/or exosomes.
  • cancer cells secretion in particular such as mucus, extracellular vesicles, oncosomes, ectosomes and/or exosomes.
  • the detection and analysis of cells secretion according to the present invention can assist early patient prognoses and determine appropriate tailored treatments.
  • the ability to monitor the disease progression over time can facilitate appropriate modification to a patient's therapy, potentially improving their prognosis and quality of life.
  • the invention also concerns a method for diagnosing a disease in a subject having or being suspected to have a disease, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group X able to react with the reactive group R by click chemistry, optionally in presence of copper; (c) detecting the cell secretion labelling, and (d) measuring and comparing the cell secretion labelling to a reference level, the cell secretion labelling level being indicative of the disease; and/or analysing and comparing the cell secretion to a biological sample of
  • the invention also concerns a method for diagnosing a disease in a subject having or being suspected to have a disease, wherein the method comprises: (a) administering to a patient a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group adapted for click chemistry to a labelling agent, or a labelling agent, (b) providing a biological sample from said patient, (c) optionally isolating the cell secretion products from the biological sample of step (a), (d) optionally contacting the sample of step (b) or the isolated cell secretion products of step (c) with a compound bearing a reactive group X able to react with the reactive group R by click chemistry, optionally in presence of copper; (e) detecting the cell secretion labelling, and (f) measuring and comparing the cell secretion labelling to a reference level, the cell secretion labelling level being indicative of the presence of the disease; and/or analysing and comparing
  • the method can allow early detection of cancerous environment. It has been found herein that assimilation of the monosaccharide compound of formula (I) or metabolite thereof, in cancer cells is different from non-cancer cells, and more particularly is higher compared to non-cancer cells (e.g. reference level or control sample). In addition, it is known that cancer cells exert more secretion such as extracellular vesicles (e.g. exosomes and/or ectosomes) and mucus that non-cancer cells.
  • extracellular vesicles e.g. exosomes and/or ectosomes
  • the present invention relates to a method for identifying or isolating cancer cell secretions or for diagnosis a cancer in a subject comprising: - implementing a method for labelling a cell secretion as described herein of a sample from said subject; - detecting the labelling, after implementing of said method for labelling, and optionally comparing the labelling to a reference level; and then - identifying cancer cell secretion and/or measuring cancer cell secretion or diagnosing a cancer based on the measuring of the label, preferably in comparison with the reference level.
  • the cancer cell secretions are identified, preferably as to diagnose a cancer, when the measuring of the label cell secretion is higher than a reference level.
  • the reference level can be obtained by measuring the label in a control sample which is a non-cancer or normal sample, in particular such as described below under the paragraph “Biological sample and reference sample”. According to the method of the present invention, in absence of labelling or detection, one can conclude that the biological sample does not comprise any cell secretion. Prognosis and response to treatment
  • the methods disclosed herein can be used to predict or monitor a response to a treatment and/or to predict the clinical outcome of a patient, in particular a patient suffering from a disease, in particular such as cancer.
  • the terms “clinical outcome” refers to the determination as to whether a subject is likely to be affected by a relapse, recurrence or death. These terms also relate to the survival, in particular the overall survival.
  • “Overall survival” as used herein refers to the time span from starting the treatment until disease specific death of the patient.
  • a poor prognosis is a poor survival, a relapse, formation of premetastatic niches, and/or metastasis.
  • the disease recurrence particularly occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the disease’s treatment by any method known by the person skilled in the art, such as surgery, antibiotherapy, radiotherapy, chemotherapy, targeted therapy, immunotherapy or any combinations thereof.
  • the cancer recurrence particularly occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after cancer treatment by any method known by the person skilled in the art, such as surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy or any combinations thereof.
  • the poor overall survival of a patient is particularly an overall survival decreased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months in comparison with the median or mean overall survival of a population of patients suffering from the same disease.
  • the invention relates to a method, preferably an in vitro or ex vivo method for predicting the clinical outcome of a subject having or being suspected to have a disease, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group adapted for click chemistry to a labelling agent or a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group X able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting the cell secretion labelling, and (e) measuring and comparing the cell secretion labelling to a reference level, the cell secretion labelling level being indicative of the clinical outcome; and/or analysing
  • the method can further comprise one or more washing steps, preferably 2 washing steps, in particular prior to step d).
  • the invention concerns a method, preferably an in vitro or ex vivo method, for predicting the clinical outcome of a subject having or being suspected to have a disease, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group adapted for click chemistry to a labelling agent or a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting and measuring the cell secretion labelling, and (e) comparing the cell secretion
  • the method can further comprise one or more washing steps, preferably 2 washing steps, in particular prior to step d).
  • the invention particularly concerns in vitro or ex vivo method for predicting the clinical outcome of a subject having or being suspected to have cancer, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group adapted for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group X able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting and measuring the cell secretion labelling, and (e) comparing the cell secretion labelling level to a
  • the method can further comprise one or more washing steps, preferably 2 washing steps, in particular prior to step d).
  • the cell secretions are cancer cell secretions.
  • the cell secretions are mucus, extracellular vesicles, oncosomes, ectosomes and/or exosomes, preferably oncosomes.
  • the reference level can be obtained by measuring the label of cell secretion in a control sample which is a non-cancer or normal sample, in particular such as described below under the paragraph “Biological sample and reference sample”.
  • the labelling ratio of the sample with respect to the reference sample is more than 1.1, 1.2, 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 labelling ratio of the sample with respect to the reference sample is more 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.
  • the reference sample is preferably a non-cancer sample.
  • the higher labelling of cell secretion in the biological sample from the patient having cancer or suspected of having a cancer in comparison to the normal sample is predictive or indicative of a poor prognosis.
  • the invention also concerns a method, preferably an in vitro or ex vivo method, for predicting a response of a subject suffering from a disease to a treatment, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting the cell secretion
  • the invention also relates to a method, preferably an ex vivo or in vitro method, for predicting or monitoring a response of a subject suffering from cancer to an anticancer treatment, wherein the method comprises: (a) contacting a biological sample from said subject with a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group for click chemistry to a labelling agent or is a labelling agent, (b) optionally isolating the cell secretion products from the biological sample of step (a), (c) optionally contacting the sample of step (a) or the isolated cell secretion products of step (b) with a compound bearing a reactive group X able to react with the reactive group R by click chemistry, optionally in presence of copper; (d) detecting and measuring the cell secretion labelling, and (e) comparing the cell secretion labelling to a reference level, the cell secretion labelling level being indicative or predictive of the responsiveness of
  • the method can further comprise one or more washing steps, preferably 2 washing steps, in particular prior to step d).
  • the level of cell secretion labelling in the biological sample is compared to a reference level.
  • a higher labelling of cell secretion is predictive of a poor response to the anticancer treatment.
  • higher measuring or “higher labelling”, it denotes that the labelling ratio of the sample with respect to the reference sample is more than 1.1, 1.2, 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.
  • the labelling ratio of the sample with respect to the reference sample is more 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.
  • the reference sample is preferably a non-cancer sample.
  • “non-responder” or “poor responder” refers to a subject who does not respond to an anti-cancer treatment, for example such as the volume of the tumour does not substantially decrease, or the symptoms of the cancer in the subject are not alleviated, or the cancer progresses, for example the volume of the tumour increases and/or the tumour generates local or distant metastasis or leads to the death of the subject.
  • non-responder is also intended a patient who shows a weak therapeutic benefit of the treatment, that is to say a shorter disease-free survival, a shorter overall survival, an increased metastasis occurrence and/or an increased tumour growth in comparison to a population of patients suffering from the same cancer and having the same treatment.
  • the compound of formula I or a metabolite thereof can be administered to a subject by any route of administration (i.e. in vivo).
  • step (a) comprises providing a biological sample from a subject which has been previously administered with a compound of formula (I) or metabolite thereof such as disclosed herein, wherein R is a reactive group adapted for click chemistry or is a labelling agent.
  • the compound bearing a reactive group able to react with the reactive group R by click chemistry can be put in contact of cell secretion in vitro or ex vivo afterwards, as detailed above for optional steps (b) and (c), for example after providing a biological sample from the subject, and optionally after having isolated cell secretion from the biological sample
  • the cell secretion products can be isolated from the biological sample of step (a) by any means, such as by filtration (e.g. with cell strainer), as to separate the cell secretion products from the secreting cells, for instance.
  • Biological sample and reference sample The methods and uses according to the invention comprise a step of characterizing a biological sample from a patient.
  • the methods and uses according to the invention may comprise an initial step of providing samples from a patient.
  • the sample can, for example, be obtained from a subject by, but not limited to, venipuncture, excretion, biopsy, resection, needle aspirate, lavage sample, scraping, surgical incision, or, any combination thereof, and the like.
  • biological samples include fluids such as blood, plasma, saliva, urine, serum, breast milk, cerebrospinal and seminal fluid samples, as well as biopsies, organs, tissues or cell samples, such as cancerous cells or tissues.
  • the sample is collected from blood or urine.
  • the sample according to the invention is a blood fluid.
  • Blood tests are easy and safe to perform and multiple samples can be taken over time, for example during a period in which a patient is treated for a disease such as cancer, for monitoring the evolution of the disease.
  • the biological sample may or may not comprise abnormal, altered, deficient, infected, damage, dysfunctional, diseased or cancerous cells.
  • the biological sample may or may not comprise tumour cells. Indeed, it is known in the art that cancer cell secretion can be disseminated through the whole subject body.
  • extracellular vesicles such as exosomes and/or ectosomes are released by cancer cells and deliver signalling molecules and genetic material and mobilize bone marrow-derived cells to colonize premetastatic niches (PMNs).
  • PMNs premetastatic niches
  • Cancer cell secretions orchestrate the establishment of a unique ecosystem or microenvironment, consisting of myriads of cells promoting incoming tumour cells to grow, spread and eventually, resist therapy. Then, in one embodiment, the sample is collected from body fluids such as blood or urine, or from a distant organ from the primary tumour, for example such as lymph nodes. Alternatively, the sample can be collected from the primary tumour.
  • the biological sample may or may not comprise infected cells.
  • the sample may be treated prior to being used (e.g., diluted, concentrated, separated, partially purified, frozen, etc.). As example, a cell enrichment sorting may be performed.
  • the sample may be fresh, frozen or fixed (e.g. formaldehyde or paraffin fixed) sample.
  • the biological sample is treated with a lysis buffer, for example such as Triton.
  • a lysis buffer for example such as Triton.
  • the biological sample is centrifuged, for example at 100- 500 g, preferably at 400 g, for a duration of 1 to 10 minutes, preferably 5 minutes, under ambient temperature (e.g. between 18°C and 25°C).
  • Such centrifugation may be performed in any methods disclosed herein, in particular so as to recover extracellular vesicles, preferably exosomes.
  • the methods according to the invention can be carried out with one or more biological samples simultaneously, using for instance a micro- well plate.
  • the microplate typically has 6, 12, 24, 48, 96, 384 or 1536 sample wells.
  • each well preferably comprises a biological sample from a subject, for example a cancer sample, a blood or urine sample or a lymph node sample.
  • Detection and/or measure of the labelling agent can be performed by any methods known in the art.
  • fluorescence plate reader for example such as provided by Biotek, Synergy Mx, Colmar, France.
  • Fluorescence quantification may be performed using fluorescence plate reader, for example such as provided by Biotek, Synergy Mx, Colmar, France.
  • the cell secretion is selected from the group consisting of mucus, membrane-bound secretory vesicles, extracellular vesicles, oncosomes, ectosomes and/or exosomes.
  • the cell secretion is an extracellular vesicle, such as ectosomes and/or exosomes.
  • the cell secretion is mucus.
  • extracellular vesicles or “EV”, or “membrane-bound secretory vesicles” refers to lipid bilayer-delimited particles that are naturally released from a cell and, unlike a cell, cannot replicate.
  • EVs range in diameter from near the size of 20-30 nanometres to as large as 10 microns or more. They carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. EVs include exosomes, ectosomes and oncosomes. Tumour-derived extracellular vesicles can be also called oncosomes. Oncosomes are particularly extracellular vesicles carrying abnormal and transforming macromolecules from a parental cancer cell, such as oncogenic proteins. The term “exosomes” relates to 30 to 100 nm vesicles secreted by a wide range of mammalian cell types, such as cancer cells.
  • Exosomes consist of a lipid bilayer membrane surrounding a small cytosol and are devoid of cellular organelles. Exosomes contain various molecular constituents of their cell of origin, including proteins and nucleic acid material. Exosomes guide the export of major types of proteins and transcription factors to the outer- cellular milieu. Such exosomal secretion of proteins impact distant cell signalling or promote a niche that sustains tumour microenvironment leading to cancer spread. Due to their presence and stability in most body fluids and resemblance of their contents to parental cells, exosomes have great potential as biopsy specimens for various diseases and cancers.
  • the cell secretion is a cancer cell secretion such as tumour- secreted exosomes (TSE) and/or oncosomes.
  • TSE tumour- secreted exosomes
  • ectosomes “microvesicles” or “MV” are generally larger vesicles than exosomes (up to ⁇ 1,000 nm in diameter), but can also be small vesicles that bud from the cell surface. They are found both in tissues and in many types of body fluids. Microvesicles may reflect the antigenic content of the cell of origin and have a role in cell signaling.
  • mucus refers to a viscid slippery secretion that is usually rich in mucins and is produced by cells or mucous membranes which it moistens and protects. It is composed of salts, desquamated cells and leukocytes. In the context of cancer, overproduction of mucus is important for the formation of tumours and can make them resistant to anticancer treatment. Cells secretion are secretion from cells of any type.
  • cell secretion can be secretion from cancer cells, neural cells, neuronal progenitor cells, immune cells, blood cells, haemopoietic cells, a cardiac cells (cardiomyocytes), pancreatic islets, hepatocytes, lung cells, pancreatic cells, gastrointestinal cells, muscle cells or renal cells.
  • cell secretions are secretions from cancer cells of any type. They can come from solid tumours or hematopoietic cancers. Cancer cells include circulating or non- circulating tumour cells. Circulating tumour cells (CTCs) are cells that have shed into the vasculature or lymphatics from a primary tumour and are carried around the body in the blood circulation.
  • CTCs Circulating tumour cells
  • the cancer cells are cells from 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, renal 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, blood, skin, pancreas, brain, liver, kidney, lung, muscle, lymphocyte, prostate, stomach, and breast cancer.
  • the cancer to be diagnosed is selected from the group consisting of: bladder, blood, colon, stomach, breast, lung, skin, and pancreas cancers.
  • the cancer cells are cells from liver cancer, cholangiocarcinoma, nasopharyngeal cancer, lung cancer, gastric cancer, spongioblastoma, prostate cancer, bladder cancer, breast cancer, colorectal cancer, melanoma, ovarian cancer, pancreatic cancer, renal cell carcinoma or stomach cancer.
  • the content or composition of such secretion may vary.
  • Cell secretion comprises a wide variety of DNA, RNA, mRNA, miRNA, proteins lipids, metabolites, hormones, cytokines, or even organelles. Such content may serve as biomarkers for a disease. Any of the methods disclosed herein can comprise a comparison to a reference level, more specifically to a control sample or reference sample.
  • the content or composition of the cell secretion of a biological sample is compared to the content or composition of the cell secretion of a biological sample of reference.
  • the reference level can be the intensity of the labelling (such as fluorescence or radioactivity) measured in a normal or healthy biological sample.
  • the normal sample can be a sample from a healthy subject or from a population of healthy subjects or from the subject himself.
  • the normal sample can also be a sample from the same patient, preferably a normal, non-tumour or healthy sample, preferably derived from the same origin.
  • the sample of reference is the closest of the sample to be studied, preferably a sample or a cell from the same origin.
  • the biological sample and the reference sample are histological matched samples.
  • a histologically matched normal sample or “matched normal sample” or “matched normal control” is meant herein a sample that corresponds to the same or similar organ, tissue or fluid as the biological sample to which it is compared.
  • a histologically matched normal sample can be a matching normal mammary tissue sample.
  • the histologically matched normal sample is for example a sample from normal bronchial mucosa.
  • the biological sample from a subject is a blood sample
  • the reference sample is a blood sample from a healthy subject or from a population of healthy subjects.
  • the “normal” sample does not comprise any cancer cell.
  • the method may comprise a previous step of providing a biological sample and a histologically matched normal sample from the subject.
  • the normal and tumour status of samples used are certified by trained pathologist. To avoid any doubt, surrounding normal tissue in proximity to the tumour is preferably excluded.
  • kits are provided for the diagnosis of a patient suffering from a disease and/or determining the presence and/or amount of cell secretion, in particular by the diagnosis methods of embodiments described above.
  • the kit may include an apparatus for collecting a sample from a patient, labels, reagents, other materials necessary to label the cells secretion, and instructions for preparing reagents and/or performing labelling of cells secretion and diagnostic.
  • the kit may be a diagnostic kit.
  • the kits are used before and/or after the subject treatment such as anti-cancer treatment.
  • the kits are used before and/or after the subject first diagnosis, for example for monitoring the evolution of the disease.
  • the components of the kits may be packaged either in suitable form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the kit comprises: (a) a compound of formula (I): or a metabolite of compound of formula (I) presenting a R group, wherein R is a reactive group for click chemistry to a labelling agent or a labelling agent, (b) optionally a compound bearing a reactive group able to react with the reactive group R by click chemistry, (c) optionally click chemistry reagent(s), and (d) optionally, a leaflet providing guidelines to use such a kit and method to evaluate the expression level of such biomarkers.
  • the invention also concerns the use of a kit as disclosed above for labelling cells secretion and/or concentrating cells secretion and/or diagnosing a disease, preferably such as cancer, neurodegenerative disease, autoimmune disease, infectious disease such as bacterial or viral infection or cardiovascular disease.
  • a disease preferably such as cancer, neurodegenerative disease, autoimmune disease, infectious disease such as bacterial or viral infection or cardiovascular disease.
  • the invention also relates to a diagnostic kit for detecting a cancerous condition in a subjection, the kit comprising a compound or composition as described herein, for the detection of secretion of cancer cells.
  • the methods and the kit according to the invention are useful to identify cell secretion by detection of labelling, particularly cancer cell secretion.
  • the invention finally relates to the use of the compound or composition described herein as labelling agent or diagnosis agent, in particular for the labelling or concentrating or diagnosis of cell secretion.
  • the present invention particularly concerns the use of a modified monosaccharide compound of formula (I) or a metabolite thereof as disclosed herein as a research tool for detecting or concentrating cell secretion, and more particularly to identify or isolate cell secretion. Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting. Examples Example 1: Synthesis of compounds Materials and methods: Thin layer chromatography was performed over Merck 60 F254 with detection by UV, and/or by charring with sulphuric acid or KMnO 4 or phosphomolybdic acid solutions. Silica gel 6040-63 Mm was used for flash column chromatography.
  • NMR spectra were taken on Bruker Avance 300 or 500 MHz spectrometers, using the residual protonated solvent as internal standard. Chemical shifts ⁇ are given in parts per million (ppm) and coupling constants are reported as Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), doublet of doublet (dd), doublet of doublet of doublet (ddd). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m). Mass spectra were taken on a Waters LCT Premier XE (ToF), with electrospray ionization in the positive (ESI+) or in the negative (ESI-) mode of detection.
  • ToF Waters LCT Premier XE
  • IR-FT spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Characteristic absorptions are reported in cm -1 . Specific optical rotations were measured at 20°C with an Anton Paar MCP 300 polarimeter in a 10-cm cell at 20°C and 589 nm. All biological and chemical reagents were of analytical or cell culture grade, obtained from commercial sources, and used without further purifications.
  • Ara-N3 was synthesized according to the following procedure: with R 1 , R 2 , and R 3 being a methyl group.
  • Cell exposure to Ara-N3 also called XN3 in the Figures
  • Cells were seeded in 24-well plates, at an initial density of 5x10 4 cells/well. Then, cells were incubated at 37°C, 5% in CO 2 during 24 h. Thereafter, the culture medium of each well was discarded, and attached cells were washed twice with 1 mL of cold Phosphate Buffer Salin (PBS ++ from Gibco), before to be exposed to Ara-N3 probe (10 mM) diluted in culture medium. Then, cells were incubated at 37°C, 5% in CO 2 for a time corresponding to either one or two doubling time depending of the experiment, such time being specific of each human cancer cell line.
  • PBS ++ cold Phosphate Buffer Salin
  • Negative control consisted in cells incubated for the same period of time in culture medium without Ara-N 3 .
  • Labelling with anti-biotin antibodies The incorporation of Ara-N 3 probe was visualized by a copper-free click chemistry using sulfo-DBCO-biotin (1 mM), followed by labelling with a mouse anti-biotin Alexa Fluor 488 antibody conjugate (0.62 mg/ml stock, Jackson ImmunoResearch, dilution used: 1/10). After incubation with Ara-N 3 , the culture media were removed, and attached cells were washed twice with 1 mL of cold PBS ++ .
  • This labelling of the cell secretion, especially mucus-like extracellular matrix may explain the results obtained with the other assays not related to microscopy, where an increase in total fluorescence in the presence of the Ara-N3 probe was observed on epithelial cancer cells.

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Abstract

L'invention concerne des composés monosaccharidiques, notamment des composés désoxy-pentose et de préférence du 5-azido-5-désoxy-D-arabinofuranose, destinés à être utilisés dans le marquage et/ou la concentration et/ou le ciblage et/ou le diagnostic de sécrétions cellulaires à partir de cellules ou de bactéries eucaryotes. Les monosaccharides peuvent être utilisés dans des procédés visant à prédire le résultat clinique ou à prédire la réponse à un traitement. De préférence encore, les monosaccharides sont marqués par chimie click. Les exemples montrent que les monosaccharides marquent des cellules cancéreuses dans une plus large mesure que les cellules normales.
EP21763340.3A 2020-08-19 2021-08-18 Composé monosaccharidique pour le marquage de la sécrétion cellulaire Pending EP4200613A1 (fr)

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US4036945A (en) 1976-05-03 1977-07-19 The Massachusetts General Hospital Composition and method for determining the size and location of myocardial infarcts
US4311688A (en) 1979-10-29 1982-01-19 Serono Laboratories Inc. Composition and method for cancer detection in humans
EP3091081A1 (fr) 2015-05-04 2016-11-09 Centre National de la Recherche Scientifique (CNRS) Procédé pour marquer spécifiquement des bactéries vivantes comprenant l'utilisation de composés de monosaccharide modifiés
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