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    • 专利摘要:
    Compound for the detection of senescent cells and their use. The invention relates to a detection compound or probe that has a high diuretic activity and is constituted by a signaling unit (fluorophore) provided with functional groups with negative charge (anionic groups). Attached to the signaling unit by direct covalent bonding is the recognition unit consisting of a saccharide. The invention also extends to the use of the compound as a senescent cell detection probe in the urine. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2733357A1
    申请号:ES201930893
    申请日:2019-10-10
    公开日:2019-11-28
    发明作者:Torres Beatriz Lozano；Barradas Juan Francisco Blandez；Mañez Ramón Martínez；Guillem Irene Galiana；Fernández Alba García；Galarza Félix Sancenón；Calatayud Mar Orzáez
    申请人:Centro De Investig Principe Felipe；Universidad Politecnica de Valencia；Centro de Investigacion Principe Felipe；
    IPC主号:
    专利说明:

    [0001]
    [0002] Compound for the detection of senescent cells and their use
    [0003]
    [0004] In the present invention a compound, also called a probe or diagnostic tool, is described for the detection of senescent cells through urine, achieving a quantification of cellular senescence. The present invention thus falls within the field of medicine and, more particularly, in diagnostic tools.
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007]
    [0008] The main role of cell senescence is the prevention of proliferation of damaged or stressed cells and their subsequent elimination. On the other hand, cellular senescence acts as a trigger for the activation of repair and regeneration processes of damaged tissues or during the embryonic process, which indicates that the presence of senescent cells can also be of benefit to the biological system. However, these tissue repair processes can become inefficient when there is a persistence of cell damage or during aging. This insufficiency allows senescent cells to accumulate on damaged tissues or organs, which promotes local inflammations, aging and destruction of tissues, the formation of tumors and even the generation of metastases. It has been shown that the selective elimination of senescent cells through cell apoptosis (senolysis) promotes the improvement of a wide variety of diseases associated with the presence of senescent cells, reversing their symptoms and improving the quality and life expectancy of individuals. . In fact, studies have been carried out in murine models where the efficacy of senolysis processes for the elimination and improvement of the symptoms of diseases such as renal dysfunction, pulmonary fibrosis, hypertrophy or sarcopenia has been corroborated. In the literature it is described that certain small molecules have therapeutic effects for multiple diseases, this pharmacological activity is based on the ability to promote senolysis. Based on these same principles, there are studies that show that senolysis can have the same efficacy for the treatment of diseases related to cell aging.
    [0009]
    [0010] Within the field of cellular senescence, it is extremely important to develop new methods to achieve the detection of senescent cells as soon as they appear in the organism, as well as at the moment they begin to accumulate. However, the vast majority of the methods described today cannot be applied for the direct detection of senescent cells and virtually none of them can be used in in vivo models . With the above, it is clear the need for the development of new systems or methods for the detection of senescent cells that can be used as diagnostic tools, and even more important, is to be able to achieve a method that allows quantifying the degree of cellular senescence and allow the follow-up of treatments with senotherapeutics in the different diseases associated with the accumulation of senescent cells.
    [0011]
    [0012] US2016229840 describes fluorogenic compounds designed in such a way that, in the event of a chemical event, compounds capable of emitting fluorescence are generated. The uses of fluorogenic compounds to monitor the presence and / or level of various analytes are also described. As opposed to document US2016229840, where probes with photochemical properties similar to those described in the present invention are exposed, the probes proposed in the present invention have diuretic characteristics that allow the measurement of analytes, in this case the detection of senescent cells, of a Non-invasive way through the urine. Said diuretic property implies that the probes are quickly expelled from the body avoiding bioaccumulation of the same and allowing the storage of the samples for later measurement if it could not be performed in situ.
    [0013]
    [0014] ES2710322 (T3) is based on compounds useful for visualizing cell senescence, its preparation and use. In particular, this invention relates to new fucose and amino-quinoline derivatives useful as senescence tracers and their preparation. Document ES2710322 (T3) employs probes for the detection of senescence consisting of two types of markers, one fluorogenic and a second radioisotope marker. In vitro senescence detection is performed by fluorescence using techniques based on flow cytometry, such techniques prevent its use for in vivo systems. On the other hand, in vivo senescence detection is performed with halogen radioisotopes. In the present invention we propose a fluorescence based in vivo senescence detection method, the simplest method where much more common and affordable measuring equipment is used, techniques that can be employed by unqualified personnel. On the other hand, though In both cases they are two non-invasive techniques, in the present invention we propose the measurement of senescence through the urine due to the intrinsic characteristics of the probes, this allows the storage of the samples for a later measurement in case that no Direct measurements of the samples can be made. Finally, compared to the probes described in document ES2710322 (T3) which present complex synthetic routes, in the present invention the synthetic route described consists of two stages, starting directly from commercial and low-cost compounds.
    [0015]
    [0016] Therefore, the present invention proposes a new tool (probe) for the non-invasive detection of cellular senescence in subjects through urine.
    [0017]
    [0018] DESCRIPTION OF THE INVENTION
    [0019]
    [0020] In a first aspect, the present invention relates to a compound characterized by comprising:
    [0021] - a signaling unit consisting of a fluorophore comprising at least one anionic group and
    [0022] - a recognition unit consisting of a saccharide,
    [0023] where the signaling unit is directly and covalently linked to the recognition unit.
    [0024]
    [0025] Direct and covalent union implies the non-existence of union groups or linkers between the signaling unit and the recognition unit. Therefore, the compound of formula:
    [0026]
    [0027]
    [0028]
    [0029]
    [0030] where R is H or an acetyl group (CH3-CO-) would be excluded from the scope of the present invention, since there is a linking group between the monosaccharide and the fluorophore.
    [0031] The term "saccharide" refers to a carbohydrate constituted by, or consisting of, at least one monosaccharide (basic sugar unit), where the monosaccharides that make up the saccharide are linked together by glycosidic bonds. In the present invention, the term "monosaccharide" includes the basic sugar unit comprising -OH groups (for example, glucose, galactose, fructose, etc.), as well as derivatives thereof. These derivatives are obtained by replacing some of the hydroxyl groups with some other functional group, or by oxidation or reduction of any of the carbon atoms of the original monosaccharide. The derivatives may be, for example, amino sugars, in which at least one hydroxyl group of the originating monosaccharide is substituted by an amino group; sugar acids, which result from the oxidation of some carbon atom of the monosaccharide of origin to a carboxyl group; deoxy sugars, in which some of the hydroxyl groups of the monosaccharide of origin are substituted by a hydrogen atom; phosphate sugars, which are phosphorylated sugars (linked to a phosphate group by ester linkage) in one of its hydroxyl groups, monosaccharides obtained by replacing at least one -OH group with a -OR ', where R' is selected from: acetyl (Ac: CH3-CO-), C1-C8 alkyl, arylalkyl, aryl and heteroaryl. In the latter case, the monosaccharide may have several or all substituted -OH groups. Preferably, the monosaccharide derivatives are those that have all their -OH substituted by -OAc. Within the monosaccharides are included the different anomeric forms thereof: a (alpha) and p (beta), as well as their Dextro (D) and Levo (L) configurations.
    [0032]
    [0033] The term "C1-C8 alkyl" refers, in the present invention, to aliphatic, linear or branched chains, having 1 to 8 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n -butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Preferably the alkyl group has between 1 and 4 carbon atoms. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido.
    [0034]
    [0035] By "arylalkyl" is meant in the present invention an aryl group attached to the rest of the molecule by an alkyl group. A non-limiting example of arylalkyl is a benzyl group.
    [0036] The term "aryl" refers in the present invention to an aromatic carbocyclic chain, having from 6 to 12 carbon atoms, being able to be single or multiple ring, in the latter case with separate and / or condensed rings. An example, non-limiting, aryl is a phenyl group.Aryl groups are, for example, but not limited to phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthracil.Aryl radicals may be optionally substituted by one or more substituents.
    [0037]
    [0038] The term "heteroaryl" refers, in the present invention, to aromatic cyclic rings (mono- or bicyclic) having between 5 and 10 links in which a carbon has been replaced by an electronegative element such as sulfur, nitrogen, or oxygen Preferably the heteroaryl group has between 5 and 6 links The aryl radicals may be optionally substituted by one or more substituents such as alkyl, halogen, hydroxyl, alkoxide, thiol, nitro, amino, acylamino, cyano, carboxylate, carboxamide groups , carboxy ester, or combinations of these groups.
    [0039]
    [0040] The term "fluorophore" refers to a molecule capable of emitting electromagnetic radiation in response to the absorption of an excitation radiation, the wavelength of the emitted radiation being different from the wavelength of the excitation radiation and where the Radiation emission persists only while the excitation radiation is maintained.This emission of fluorophore radiation of the invention originates at wavelengths that do not overlap with the self emission from senescent cells existing in the biological samples.
    [0041]
    [0042] The chemical bond between the signaling unit and the recognition unit is a covalent bond, preferably through an N, S, P or O atom of the signaling unit and the saccharide anomeric C atom, where the OH group of the Anomeric carbon is replaced by fluorophore. The signaling unit consisting of a fluorophore has at least one anionic group (also called a diuretic unit), which is responsible for carrying out the diuretic function and increasing the solubility of the compound so that it can be excreted in the urine. The anionic group can be selected from, for example and not limited to, carboxylate group (-CO2 "), sulphonate (-SO3"), sulfate (-SO4 "), halide (F-, Cl", Br- and I- ) or any anion capable of interacting with the Na + -K + -2Cl- co-transporter proteins that are located in the cells of the ascending limbs of Henle's handles. In a preferred embodiment, the anionic groups bound to the fluorophore are sulfonate and / or groups carboxylates. In a more preferred embodiment, the signaling unit comprises two anionic groups. More preferably two sulfonate groups.
    [0043]
    [0044] Preferably, the fluorophore that forms the signaling unit is based on a cyanine structure having anionic groups covalently linked to confer diuretic properties. More preferably, the fluorophore has a structure (formula (I)) as defined below:
    [0045]
    [0046]
    [0047]
    [0048] where A 1 and A2 are independently aryl or heteroaryl groups (fused to the pyrrolinium ring, as the structure shows),
    [0049] p and q are independently an integer that varies between 1 and 4,
    [0050] R1 and R2 are independently a group - (CH2) n-Ra, where:
    [0051] Ra is an anion that is selected from the list consisting of: -SO3- -CO2-, -SO4-, halide,
    [0052] n is an integer that varies between 0 and 8,
    [0053] X is O, N, P or S and
    [0054] R3, R4, R5, R 6 are independently selected from: C1-C8 alkyl, aryl and heteroaryl,
    [0055] represents the point of covalent attachment with a carbon of a monosaccharide of the recognition unit.
    [0056]
    [0057] The terms C1-C8 alkyl, aryl and heteroaryl are as previously defined.
    [0058]
    [0059] In a preferred embodiment of the fluorophore, A1 and A2 are independently phenyl.
    [0060]
    [0061] In another preferred embodiment of the fluorophore, p and q are independently 1.
    [0062]
    [0063] In another preferred embodiment of the fluorophore, R1 and R2 are independently a group - (CH2) n -SO3 "or - (CH2) n -CO2", n between 1 and 8, more preferably n is 4.
    [0064] In another preferred embodiment of the fluorophore, X is O.
    [0065]
    [0066] In a preferred embodiment of the fluorophore, R3, R4, R5, R 6 are independently methyl.
    [0067]
    [0068] In another preferred embodiment, A1 and A2 are independently phenyl; p and q are independently 1 and X is O.
    [0069]
    [0070] In another preferred embodiment, A1 and A2 are independently phenyl; p and q are independently 1; X is O and R1 and R2 are independently - (CH2) 4 -SO3 "or - (CH2) 4 -CO2.
    [0071]
    [0072] In a preferred embodiment, the fluorophore has the following formula:
    [0073]
    [0074]
    [0075]
    [0076]
    [0077] Preferably, the recognition unit is a saccharide that comprises, or consists of, between 1 to 100 monosaccharides.
    [0078]
    [0079] In a more preferred embodiment, the saccharide comprises, or consists of, between 1 and 50 monosaccharides or, even more preferably, between 1 and 20 monosaccharides.
    [0080]
    [0081] In another preferred embodiment, 60 to 95% of the monosaccharides comprising the saccharide are galactose, fucose and / or derivatives thereof obtained by replacing at least one -OH group, as explained above in the definition of monosaccharide More preferably, from 60 to 95% of the monosaccharides comprising the saccharide are galactose, fucose and / or derivatives thereof where all of its OH have been substituted by -OAc.
    [0082]
    [0083] In another preferred embodiment, the monosaccharides comprising the saccharide are galactose, fucose and / or derivatives thereof obtained by substitution of at least one -OH group, as explained above in the definition of monosaccharide. More preferably, the monosaccharides comprising the saccharide are galactose, fucose and / or derivatives thereof where all of their OH have been substituted by -OAc.
    [0084]
    [0085] In another preferred embodiment, the saccharide consists of a monosaccharide, preferably this is galactose, fucose or a derivative of any of them where all its OH have been substituted by -OAc.
    [0086]
    [0087] Preferably, when reference is made in the present invention to galactose, this is p-D-galactose and when reference is made to fucose, this is preferably a-D fucose.
    [0088]
    [0089] In a more preferred embodiment, the compound has the following formula:
    [0090]
    [0091]
    [0092]
    [0093]
    [0094] where R is H or an acetyl group (CH3-CO-).
    [0095]
    [0096] Senescent cells are characterized by an increase in the number and size of lysosomes that causes overexpression of lysosomal hydrolase enzymes, the most characteristic of which are the overexpression of activity of the lysosomal enzyme p-galactosidase known as (SA-p gal ) and overexpression of lysosomal a-fucosidase enzyme activity known as (SA-a-fuc).
    [0097]
    [0098] In particular, in the present invention, the activation of the compound occurs due to the overactivity of the lysosomal enzyme, in the previous model it would be produced by the overactivity of p-galactosidase (SA-p-gal) associated with the presence of cellular senescence . This activity carries out the hydrolysis between the signaling unit and the recognition of the compound of the present invention. The disconnection of the recognition unit causes the activation of the signaling unit. This activation is reflected by the appearance of a marked fluorescence emission. Finally, the anionic groups that are attached to the signaling unit have a high diuretic activity that allows the expulsion of the compound in the urine. When the diuretic unit is directly attached to the signaling unit, it allows the expulsion of both the activated and deactivated compound, however, only the activated compound has a fluorescence signal.
    [0099]
    [0100] The anionic groups (sulfonates or carboxylates preferably) are used as triggers of diuretic activity by interacting with the Na--K- 2 C-co-transporting proteins that are located in the cells of the ascending limbs of Henle's handles.
    [0101]
    [0102] These anionic groups bind at the existing active sites for Cl "blocking the co-transport system of the protein. In order for the functional groups to interact in the active centers of the proteins, these functional groups must present a geometry and a Electrostatic potential similar to that of the analyte, which is why the presence of anionic groups plays a major role in the diuretic process.
    [0103]
    [0104] The anionic groups present in the diuretic unit of the compound bind to the Cl "recognition positions of the co-transport proteins and cause the high diuretic activity of the compound.
    [0105]
    [0106] The compound of the present invention is therefore an easily synthesized molecule that is initially in an "off" form. This compound should be administered in a simple way undergoing a transformation in the presence of senescent cells that allows its detection by fluorescence both in the urinary system and after urination of the urine. The fluorescence intensity observed in urine samples is proportional to the amount of senescent cells, which allows quantification and monitoring in the number of senescent cells. In addition, a small amount of the activated compound accumulates in those areas where activation occurs (where senescent cells are located), so, due to the large fluorescence emission that the signaling unit presents, this small amount it is enough to locate the area that has cells immersed in senescence processes
    [0107]
    [0108] With these characteristics, the proposed probes allow the detection of senescent cells in vivo through urine fluorescence measurements both before and after urination, giving rise to a non-invasive and quantitative method for the determination of cellular senescence, all This allows its use for different applications of great interest such as:
    [0109] - Its use in the follow-up of chemotherapy treatments. Some chemotherapy treatments are based on the use of senescence inducers or the use of other medical protocols in order to carry out the transformation of tumor cells into senescent cells and achieve the detection of cell proliferation. Therefore, the compound of the invention can be used to monitor the formation of senescent cells in vivo after the use of chemotherapy, allowing the quantification of cellular senescence in a non-invasive manner through urine fluorescence measurements.
    [0110] - They can be used to obtain information in the diagnosis of diseases related to aging where conventional treatments are based on the use of senolytics. With this new tool, the elimination of these senescent cells can be monitored with the decrease in fluorescence emission obtained in urine measurements. - Finally, the variation of the recognition unit would allow the use of the same protocol for the detection and monitoring of other diseases or pathologies.
    [0111]
    [0112] The term "in vivo" detection in the present invention refers to a detection through urine samples either in the urinary system or after urination, carried out after administration of the compound of the invention. In the present invention, the term "in vivo" is used to indicate that biopsy or removal of damaged areas is not necessary, unlike what is required by the vast majority of described methods that require tissue or cell extractions to determine the presence of cellular senescence in vitro.
    [0113] Another aspect of the invention relates to a composition comprising the compound of the invention and a pharmaceutically acceptable carrier.
    [0114]
    [0115] In a preferred embodiment, the composition is a pharmaceutical composition.
    [0116] The "vehicle" is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds and allow a better dosage and administration or to give consistency and form to the composition. Therefore, the vehicle is a substance that is capable of allow a better dosage and administration or give consistency and form to the composition. The "pharmaceutically acceptable carrier" must allow the activity of the compound, be compatible with it and not cause damage to the organisms to which it is administered.
    [0117]
    [0118] The composition of the invention can be administered in any of the known ways of administration. In a preferred embodiment, the composition is configured to be administered intravenously, intraperitoneally or orally. In the latter case, the compound can be coated with a polymer that releases its contents past the stomach. Even more preferably, the composition is configured to be injected intravenously or intraperitoneally.
    [0119]
    [0120] Another aspect of the invention relates to the use of the compound of the invention, or of the composition comprising it, as a probe for the detection of senescent cells in a urine sample where said compound has previously been administered.
    [0121]
    [0122] The terms "probe" or "diagnostic tool" are used as synonyms in the present invention and refer to a chemical compound capable of selectively detecting cellular senescence. The probe of the invention also has the following characteristics:
    [0123] - a minimum or zero toxicity;
    [0124] - It presents a decrease in the fluorescence signal, or does not show fluorescence, compared to the unmodified signaling unit (fluorophore) until the moment in which the link rupture occurs in the presence of senescent cells due to a high activity of p -galactosidase, a-fucoside or other lysosomal hydrolase;
    [0125] - The probe remains stable in its inactivated form (signaling unit and recognition unit attached) until it is in the presence of senescent cells;
    [0126] - The probe retains most of the physical and chemical characteristics that allow its use in a manner similar to that of the signaling unit (fluorophore).
    [0127]
    [0128] Senescent cells are cells that have the arrested cell cycle, which prevents normal cell activity and cell division. These cells have a flattened morphology whose cytoplasm has increased the number and size of vacuoles. Senescent cells have a distinctive phenotype and a modification of their genetic expression that allows their identification. Among the tests commonly used for identification, the most important is the determination of the overactivity of the p-galactosidase (p-gal) enzyme associated with senescence.
    [0129]
    [0130] For use as a probe, the compound can be administered to a subject in any of the known ways of administration; preferably, intravenously, intraperitoneally or orally.
    [0131]
    [0132] The probe has diuretic properties (diuretic probe) so, once administered, this probe is cleaved in the presence of senescent cells so that the signaling unit is excreted in the urine and can be detected by fluorescence, revealing the existence of cells senescent Then, for this purpose, the compound of the invention can be used as a diuretic probe (probe with diuretic properties), whose active form (once it is cleaved into the two units that comprise it) can be visualized by fluorescence in excreted urine, revealing the existence of cellular senescence. Similarly, when the probe is not activated, the diuretic unit carries out its elimination, in this case there is no fluorescence in the urine. This implies that the probe does not accumulate in the body and is rapidly expelled via the urinary tract.
    [0133]
    [0134] The use of the probe for the detection of senescent cells through urine allows a quantitative study of the degree of cellular senescence present in the subject to be carried out, in addition to obtaining information on the possible presence of diseases of the subject derived from accumulation of said cells.
    [0135]
    [0136] Another aspect of the invention relates to the compound of the invention for use in a method of detecting senescent cells through urine. More concretely, for or in, a method of detecting senescent cells in a urine sample of a subject to which said compound has previously been administered.
    [0137]
    [0138] Another aspect of the invention relates to a method of detection of senescent cells, which comprises the following steps:
    [0139] - administration of an effective amount of the compound of the invention to a subject, - obtaining a urine sample after administration of the compound,
    [0140] - obtaining fluorescence data from the urine sample obtained.
    [0141]
    [0142] In the present invention, the term "effective amount" refers to that amount of the compound that when administered, is sufficient to be detected in the urine once it has been hydrolyzed in the body.
    [0143]
    [0144] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0145]
    [0146] BRIEF DESCRIPTION OF THE FIGURES
    [0147]
    [0148] FIG. 1. General structure of a probe where you can see the different units that make it up: signaling unit that includes diuretic units or groups and recognition unit.
    [0149]
    [0150] FIG. 2. Structure of Cy7-Gal, one of the possible probes (compounds of the present invention), where the different groups are shown each with an individual function.
    [0151]
    [0152] FIG. 3. (ad) Images of confocal microscopy for 4T1 cells. (a, b) images obtained for normal 4T1 cells. (c, d) Images of 4T1 cells treated with Palbociclib to induce cellular senescence. (a, c) Images of control and senescent cells in the absence of the Cy7-Gal probe. (b, d) Control and senescent cells after being incubated 30 min in the presence of Cy7-Gal. e) Quantification of intensity in fluorescence emission for control 4T1 cells and treated with Palbociclib (senescent) incubated in the absence or in the presence of the Cy7-Gal probe, f) Viability of control and senescent 4T1 cells after incubation with Cy7-Gal at different concentrations after 24 h
    [0153]
    [0154] FIG. 4. a) live in images obtained by the technique of forming images in vivo (IVIS, from the English "in vivo imaging system") where mice were injected with tumor cells 4T1 (mouse mammary carcinoma). On the left, a treatment with Palbociclib was carried out to obtain the transformation of the tumor cells into senescent cells and to stop the growth of the tumor, while in the right (vehicle) said treatment with Palbociclib was not carried out maintaining the 4T1 cancer cells. b) Quantification of Cy7 fluorophore in the bladder as a function of time. c) Quantification of fluorescence in the urine with the IVIS technique after the metabolization of the Cy7-Gal probe in the body. The mice urinated after exiting anesthesia due to the diuretic properties of the Cy7-Gal probe.
    [0155]
    [0156] FIG. 5. a) Ex vivo images of tumors from 4T1 cells extracted after sacrificing the mice of the different groups. b) Quantification of the fluorescence of Cy7 in the extracted tumors.
    [0157]
    [0158] FIG. 6. a) Images obtained by the IVIS technique where it was injected with 4T1 cells (mouse breast carcinoma). b) Quantification of the fluorescence signal in the bladder at different times and concentrations of Palbociclib. c) Enlargement of the fluorescence signal at 15 min for the different treatment groups.
    [0159]
    [0160] FIG. 7. a) IVIS images of the urine obtained for the different groups, from left to right the columns correspond to the groups: not treated with Palbociclib, 10 mg / Kg of Palbociclib, 50 mg / Kg Palbociclib, 100 mg / Kg, all of them treated with the probe, and finally, 100mg / kg Palbociclib not treated with the Cy7-Gal probe. b) Fluorescence quantification for urine samples from different groups with the IVIS technique. c) Fluorescence measurements obtained for the urine collected from the different groups. d) Fluorimetric quantification of the urine for the different groups.
    [0161]
    [0162] FIG. 8. a) Ex vivo images of tumors from 4T1 cells extracted after sacrificing different groups later staining with X-gal, commercial marker used to corroborate the existence of cellular senescence, being possible to carry out a semi-quantitative determination based on the blue-green coloration acquired by the tumor. From left to right, tumors of the group not treated with Palbociclib and the tumors obtained from the groups treated with concentrations of 10 mg / kg, 50 mg / kg and 100 mg / kg of Palbociclib are observed. b) Quantification by IVIS of the fluorescence signal for the different tumors shown above. c) Average quantification of fluorescence corresponding to the hydrolyzed probe of the tumors extracted in each group. d) Quantification by IVIS of the most representative tumors and organs for the different groups, showing that only signal is observed in senescent tumors without signal being observed in the different organs.
    [0163]
    [0164] FIG. 9. a) Immunohistochemical quantification of the degree of senescence by staining the proliferation biomarker Ki67. b) Quantification of the Ki67 biomarker that shows a drop in cell proliferation with the increase in the concentration of Palbociclib used.
    [0165]
    [0166] EXAMPLES
    [0167]
    [0168] Next, the invention will be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    [0169]
    [0170] Example 1: Synthesis of the Cy7-Gal probe:
    [0171]
    [0172] The Cy7-Gal probe was prepared according to the following reaction scheme: to
    [0173]
    [0174]
    [0175]
    [0176]
    [0177] Materials and methods:
    [0178] The compound 5-Formylsalicylaldehyde was purchased from Tokyo Chemical Industry Co., Ltd. (TCI) while a-acetobromogalactose, potassium carbonate, the inert salt of 1- (4-sulfobutyl) -2,3,3- trimethylindolium), sodium acetate and acetic anhydride were purchased from Sigma-Aldrich. The anhydrous acetonitrile was purchased from Acros Organics B.V.B.A. Saline phosphate buffer (PBS) was purchased from Sharlab S.L., Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. Palbociclib was acquired from Selleckchem. Transparent 96-plate and cellTiter-Glo (R) Luminnescent Cell plates for cell viability were obtained at Promega. Senescence beta-Galactosidase KIT for X-gal staining was purchased from Werfrem. Anti-Ki67 antibody (ab833) was obtained from Abcam. Recombined p-Galactosidase-1 / GLB1 Human protein (Human p-Gal) was obtained from the R&D system a biotechne brand. 4T1 (mouse breast carcinoma) cells were purchased from the American Type Culture Collection (AATC). BALB / cByJ mouse females were purchased from Charles River laboratories, France.
    [0179] A tripletof T5600 mass spectrometer (ABSciex, USA) was used for mass spectrometry. In the case of Nuclear Magnetic Resonance Spectroscopy (NMR) for the characterization of 1H and 13C, a Bruker FT-NMR Advance 400 (Ettlingen, Germany) spectrometer 300K was used using tetramethylsilane (TMS) as the internal standard. Fluorescence spectroscopy measurements were performed with a JASCO spectrofluorometer FP-8500. While the images of Confocal fluorescence of 4T1 cell cultures were acquired in a Leica TCS SP8 AOBS microscope and analyzed using Image software. On the other hand, the IVIS images were acquired in an IVIS® spectrum CT imaging equipment and analyzed using the Caliper Life Sciences live imaging software for both in vivo samples and in the case of organs and tumors ex vivo. The luminescence measurements for the feasibility tests were performed on a VICTOR Multilabel Plate Reader (Pelkin Elmer)
    [0180]
    [0181] Synthesis and characterization:
    [0182] The reagents 5-Formylsalicylaldehyde (75 mg, 0.5 mmol), a-acetobromogalactose (607 mg, 1.5 mmol) and potassium carbonate (400 mg, 4 mmol) were introduced into a round bottom flask under an argon atmosphere and dissolved in 30 mL of anhydrous acetonitrile. The reaction mixture was heated at 70 ° C with stirring for 4 h. maintaining the inert atmosphere. After completing the reaction, the solvent was evaporated by reduced pressure. The reaction crude was purified using a silica gel column and a hexane-ethyl acetate mixture (2: 1 v / v) as eluent. Product 1 was obtained as a light greenish oil (230 mg, 0.46 mmol, 92.6% yield).
    [0183]
    [0184] In a second stage, the product 1 obtained in the previous step (90 mg, 0.17 mmol) was introduced together with 1- (4-sulfobutyl) -2,3,3-trimethylindolium salt (100 mg, 0.35 mmol) in a tube Schlenk under inert conditions. The solids were dissolved in 2 mL of acetic anhydride. The reaction is stirred at 70 ° C for 4 h. Under inert atmosphere. After that, the solvent is evaporated under reduced pressure. The reaction crude is purified by reverse phase chromatography column using as eluent a dichloromethane: methanol mixture (10: 1 v / v) to obtain Cy7-Gal as a reddish brown solid (120 mg, 0.116 mmol, 68.1% yield) . 1H NMR (400 MHz, CDCta) 5 = 7.72-7.62 (m, 1H), 7.53 (dd, J = 7.5; 1.6 Hz, 1H), 7.49-7.43 (m, 2H), 7.42-7.32 (m, 2H), 7.00 (d, J = 7.3 Hz, 2H), 6.79 (t, J = 7.2 Hz, 1H) , 6.69 (t, J = 7.3 Hz, 1H), 6.37 (d, J = 1.7 Hz, 1H), 5.69 (d, J = 8.3 Hz, 1H), 5 , 49 (d, J = 1.3 Hz, 1H), 5.42 (dd, J = 3.4; 0.9 Hz, 1H), 5.34-5.32 (m, 2H), 5, 24 (s, 1H), 5.07 (dd, J = 10.4; 3.4 Hz, 1H), 4.17-4.04 (m, 4H), 3.48 (s, 4H), 2 , 91 (s, 4H), 2.03 (s, 12H), 1.93 (s, 4H), 1.82 (s, 4H), 1.58 (s, 12H).
    [0185] 1C-NMR (101 MHz, DMSO) 5 = 169.98 (1C), 169.88 (1C), 169.83 (1C), 169.55 (1C), 169.53 (1C), 169.51 ( 1C), 160.25 (1C), 160.01 (1C), 158.49 (1C), 142.26 (4C), 135.71 (1C), 131.03 (1C), 130.85 (1C) ), 129.31 (2C), 129.10 (1C), 127.74 (2C), 126.92 (2C), 122.89 (1C), 121.01 (2C), 115.67 (2C) , 97.17 (1C), 73.73 (1C), 71.08 (1C), 68.35 (1C), 65.90 (1C), 63.53 (2C), 63.38 (2C), 61.51 (1C), 49.02 (2C), 36.20 (2C), 28.24 (2C), 24.39 (1C), 24.26 (1C), 24.09 (1C), 23.68 (1C), 20.53 (1C), 20.48 (1C), 20.34 (1C), 20.25 ( 1 C). HRMS (high resolution mass spectrometry): Calculated for C52H63N2O16S2 (M + H +) 1035.3619 m / z; found 1035.3606 m / z (M + H +), 1051.3546 (M + H2O-2H +).
    [0186]
    [0187] Confocal microscopy:
    [0188]
    [0189] 4T1 mouse breast carcinoma cells were cultured in DMEM supplemented with 10% FBS and incubated in 20% O2 and 5% CO2 at 37 ° C. The cells were incubated with 5 pM of Palbociclib in DMEM for a week to induce the senescence process. Subsequently, the cells were seeded in a transparent flat bottom 96-well plate with a density of 6000 cells for the senescent and 4000 in control (without previous treatment with Palbociclib). After 24 hours the cells were incubated with different concentrations of the Cy7-Gal probe and its fluorophore (Cy7) and the confocal microscopy images were acquired after 30 min incubation. An increase in fluorescence emission greater than 10 times is observed between senescent cells treated with the Cy7-Gal probe and untreated senescent cells (autofluorescence signal). This increase in fluorescence emission is slightly higher between senescent cells and control cells when both have been incubated in the presence of the Cy7-Gal probe, thereby demonstrating the high selectivity of the probe in the detection of senescent cells.
    [0190]
    [0191] Fluorescence spectroscopy:
    [0192]
    [0193] Urine fluorescence measurements were carried out by taking 5pL of urine and diluting it in distilled water to 100 pL. Samples were obtained by exciting at 535 nm and collecting the maximum absorption at 56 nm. A clear correlation can be observed between the degree of senescence and the intensity of fluorescence emitted by the urine corresponding to the presence of the free Cy7 fluorophore.
    [0194]
    [0195] Feasibility tests:
    [0196]
    [0197] 4T1 mouse breast carcinoma cells were cultured in DMEM supplemented with 10% FBS and incubated in 20% O2 and 5% CO2 at 37 ° C. To obtain cellular senescence, the cells were incubated with 5 pM Palbociclib in DMEM for one week. Subsequently, the cells were seeded in a transparent flat bottom 96-well plate with a density of 6000 cells for senescent and 4000 in control. After 24 h the cells were treated with different concentrations of the Cy7-Gal probe and its fluorophore (Cy7). Viability measurements were obtained after 24 h of incubation using CellTiter-GLO Luminescent Cell Viability Assay as developer. Feasibility tests showed that both the Cy7-Gal probe and its Cy7 fluorophore have minimal toxicity even at much longer exposure times than those used in the in vivo tests .
    [0198]
    [0199] In vivo trials :
    [0200]
    [0201] The mice were kept at the Prince Felipe Research Center (CIPF) following the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). All processes applied to animals were approved by the Technical Research Committee for animal welfare (CEIyBA). 4T1 breast tumors were generated using 4T1 cells: The cells were grown in DMEM supplemented with 10% FBS and streptomycin penicillin. To generate breast tumor formation, the cells were trypsinized and counted with an automated LUNA ™ cell counter and injected subcutaneously into the left breast of female BALB / CBY mice aged 28 to 34 weeks at a concentration of 0.5-10.6 cells in a volume of 100 jl. The tumor volume was measured every two days with a caliper and was calculated as V = (ax b2) / 2 where a is the longest and b is the shortest of two perpendicular diameters. Palbociclib was administered by oral tube daily for 7 days at different concentrations of 10 mg / kg, 50 mg / kg and 100 mg / kg dissolved in 50 mM sodium lactate at pH 5 in order to induce senescence. Subsequently, the Cy7-Gal probe was administered at a concentration of 23.3 mg / ml in a volume of 100 µl. The mice were monitored for 15 minutes post-injection of the probe into the IVIS® equipment following the accumulation of Cy7 fluorophore in the bladder after hydrolysis of the Cy7-Gal probe in the presence of senescent cells. The mice urinated after exiting anesthesia, at which time both the Cy7-Gal probe and the Cy7 fluorophore were expelled from the body favored by the presence of the diuretic unit. The fluorescence of the urine samples for the different groups of mice was obtained directly using the IVIS technique and subsequently by fluorescence spectroscopy, confirming the correspondence between the fluorescence emission of the urine samples and the degree of induced cell senescence to the different subjects, as well as the possible detection of the probe in the urinary tract allowing in vivo measurement of cellular senescence.
    [0202] Subsequently, the mice were sacrificed and their organs removed quickly. To corroborate the presence of cellular senescence, the tumors were stained with the commercial kit X-gal following the protocol described for this purpose, said staining allows a semi-quantification of senescence depending on the coloration, in the same way the Immunohistochemical staining of Ki67, proliferation biomarker. Fluorescence measurement in organs and tumors for different groups of mice was performed using the IVIS technique.
    权利要求:
    Claims (17)
    [1]
    1. Compound characterized by comprising:
    - a signaling unit consisting of a fluorophore comprising at least one anionic group and
    - a recognition unit consisting of a saccharide,
    where the signaling unit is directly and covalently linked to the recognition unit.
    [2]
    2. A compound according to claim 1, wherein the saccharide comprises between 1 and 50 monosaccharides.
    [3]
    3. A compound according to claim 2, wherein the saccharide comprises between 1 and 20 monosaccharides.
    [4]
    4. Compound according to any of the preceding claims, wherein between 60 and 95% of the monosaccharide units are galactose, fucose and / or derivatives thereof.
    [5]
    5. Compound, according to any of the preceding claims 1 to 3, the monosaccharides are galactose, fucose and / or derivatives thereof.
    [6]
    6. A compound according to any one of claims 1 to 5, wherein the signaling unit comprises two anionic groups.
    [7]
    7. A compound according to any one of claims 1 to 6, wherein the anionic groups are sulfonates, carboxylates, sulfates and / or halides groups.
    [8]
    8. A compound according to any one of claims 1 to 7, wherein the fluorophore has a structure as defined below:

    [9]
    9. Compound according to claim 8, wherein A1 and A2 are independently phenyl.
    [10]
    10. Compound according to claim 8 or 9, wherein p and q are independently 1.
    [11]
    11. Compound according to any one of claims 8 to 10, wherein R1 and R2 are independently a group - (CH2) n-SO3- or - (CH2) n-CO2-, where n is an integer that varies between 0 and 8 .
    [12]
    12. Compound according to any of claims 8 to 11, wherein X is O.
    [13]
    13. Compound according to any of claims 8 to 12, wherein R3, R4, R5, R6 are independently methyl.
    [14]
    14. A compound according to claim 8, wherein the fluorophore has the following formula:

    [15]
    15. Compound according to claim 8, which has the following formula:

    [16]
    16. Composition comprising the compound described in any one of the preceding claims 1 to 15 and a pharmaceutically acceptable carrier
    [17]
    17. Use of the compound described in any one of claims 1 to 15, or of the composition described in claim 16, as a probe for the detection of senescent cells in a urine sample of a subject to which said compound has previously been administered .
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    同族专利:
    公开号 | 公开日
    ES2733357B2|2020-03-30|
    WO2021069773A1|2021-04-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2012123916A2|2011-03-15|2012-09-20|Ramot At Tel-Aviv University Ltd.|Activatable fluorogenic compounds and uses thereof as near infrared probes|
    WO2014032737A1|2012-08-28|2014-03-06|Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet|Senescence tracers|
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    PCT/ES2020/070547| WO2021069773A1|2019-10-10|2020-09-11|Compound for detecting senescent cells and use thereof|
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