EP1865974A1 - Combination consisting of a histological or cytological fixing solution and of one or more photoactivatable compounds of the family of quinones, in particular, hypericin, hypocrellin a and hyprcrellin b - Google Patents

Combination consisting of a histological or cytological fixing solution and of one or more photoactivatable compounds of the family of quinones, in particular, hypericin, hypocrellin a and hyprcrellin b

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Publication number
EP1865974A1
EP1865974A1 EP06743663A EP06743663A EP1865974A1 EP 1865974 A1 EP1865974 A1 EP 1865974A1 EP 06743663 A EP06743663 A EP 06743663A EP 06743663 A EP06743663 A EP 06743663A EP 1865974 A1 EP1865974 A1 EP 1865974A1
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EP
European Patent Office
Prior art keywords
cells
tissue
hypericin
sample
solution
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.)
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EP06743663A
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German (de)
French (fr)
Inventor
Peltier Eric
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Maclip SARL
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Novacyt SA
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Publication of EP1865974A1 publication Critical patent/EP1865974A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo

Definitions

  • the invention relates to the combination, in a solution or kit, of a histological or cytological fixator, and one or more photoactivatable compounds of the family of quinones.
  • the invention also relates to a method for labeling cells or tissues with one or more photoactivatable compounds of the quinone family, wherein the cells or tissues contacted with a histological or cytological fixator are labeled, simultaneously or afterwards, with the photoactivatable compounds of the family of quinones.
  • Photoactivatable compounds of the quinone family include, in particular, polycyclic quinones such as hypericin, hypocrelline A or hypocrelline B, or derivatives thereof.
  • “Derivative” means a chemically modified compound in which the modification is considered routine by the ordinary skill chemist, such as an ester or amide of an acid, protecting groups, such as a benzyl group for a alcohol or a thiol, and a tert-butoxycarbonyl group for an amine.
  • hypocrelline A (1H-Cyclohepta [ghi] perylene-5,11-dione, 1-acetyl-2,3-dihydro-2,6,12-trihydroxy-4,8,9,13-tetramethoxy-2- methyl-, cis-) and hypocrelline B
  • hypocrellin A hypocrellin B
  • the absorption and fluorescence emission maxima, measured in methanol, are respectively 463 nm and 600 nm for hypocrelline A, and 459 nm and
  • Hypericin has the chemical formula 1, 3,4,6,8,13-Hexahydroxy-10,11-dimethylphenanthro (1,10,9,8, opqra) perylene-7,14-dione:
  • Hypericin is a natural pigment isolated from plants of the genus Hypericum, which is a potent selective inhibitor of protein kinase C.
  • Hypericin has a variety of pharmacological properties ranging from antibacterial or antiviral activity to antineoplastic activity and induction of apoptosis. Hypericin has long been known for its effects photosensitizers and as part of the composition of an antidepressant (St. John's Wort).
  • Hypericin infused into the bladder has been shown to accumulate selectively in urothelial carcinomatous cells. This characteristic, associated with its photosensitizing and fluorescent properties, led several teams to propose a diagnosis of bladder cancer by fluorescence cytology ex vivo (Olivo et al., 2003a, Olivo et al., 2003b, Pytel and Schmeller, 2002). The use of hypericin as a photodynamic treatment of carcinomatous lesions of the bladder has also been proposed (Kamuhabwa et al., 2004). These methods involve in vivo infusion of hypericin into the patient's bladder.
  • hypericin is a lipophilic molecule that can be incorporated into the lipid bilayer of cell membranes.
  • the exact integration mechanisms are not known but it is assumed that this integration is mediated by active transport. So far, hypericin has only been used on living cells.
  • hypericin labeling on living cells, for example by culturing in vitro cells in a solution containing hypericin, or by hypericin extemporaneously in contact with freshly harvested cells or tissues, so that hypericin can be incorporated by living cells.
  • hypericin mixed with a fixative
  • hypericin is incorporated very rapidly by tumor cells suspended in the fixative / hypericin mixture, even though the cells have been killed by the contact with the fixer.
  • Fixation is an operation intended to kill the cells in order to preserve them, as much as possible, in the state they were during life.
  • fault fixation there is a risk of autolysis.
  • the best fixators are those which, while acting rapidly, produce as little as possible of secondary modifications or artifices likely to give a very false idea of the internal morphology of the cells.
  • the invention therefore relates to a solution comprising a histological or cytological fixator and one or more photoactivatable compounds of the quinone family.
  • the photoactivatable compound (s) of the family of quinones are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B. More preferably, said composition comprises, or consists of, a histological or cytological fixator and hypericin.
  • cytological fixator or “histological or cytological fixation solution” is meant a solution commonly used in histological or cytological analysis to achieve the fixation of cells or tissues.
  • cytological fixatives include fixatives based on ethyl alcohol, methyl alcohol, or polyethylene glycol (PEG).
  • histological fixatives include formaldehyde-based fixatives, such as a 10% pure formalin solution ("pure formalin” corresponding to the 40% aqueous formaldehyde solution, generally sold commercially as “formalin””), A formalin-NaCl solution (constituted for example by mixing 10 ml of pure formalin, 0.9 g of NaCl and 90 ml of water), a solution of formaldehyde - acetic acid (for example 100 ml of pure formalin, 50 ml of glacial acetic acid and 850 ml of water ), alcoholic liquor liquor (or Duboscq Brasil, for example 250 ml of pure formalin, 70 ml of glacial acetic acid, 5 g of picric acid and 680 ml of 70 ° ethanol).
  • formaldehyde-based fixatives such as a 10% pure formalin solution (“pure formalin” corresponding to the 40% aqueous formaldehyde solution, generally sold commercial
  • fixatives include alcohol-formaldehyde-acetic acid fixative (20 ml of pure formalin, 50 ml of glacial acetic acid, 750 ml of absolute ethyl alcohol, and 180 ml of water), or fixatives. based on zinc chloride (10% formalin, 10-50 g / l zinc chloride, and 5% acetic acid or NaCl 9 g / l, optionally 75% absolute alcohol).
  • the solution according to the invention advantageously comprises 1 to 20 micromoles / ml, preferably 1 to 15 micromoles / ml, preferably 1 to 10 micromoles / ml, and more preferably 1 to 2 micromoles / ml of photoactivatable compounds of the family of quinones.
  • the invention also relates to a kit comprising a histological or cytological fixator and one or more photoactivatable compounds of the quinone family.
  • the photoactivatable compound (s) of the family of quinones are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B.
  • the kit according to the invention comprises a histological fixator or cytological and hypericin.
  • the kit may optionally further comprise an appropriate wash buffer, a user's manual or any other suitable solution or device.
  • the invention also relates to a method for ex vivo labeling of cells or tissue with one or more photoactivatable compounds of the quinone family.
  • the method comprises the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising one or more photoactivatable compounds of the quinone family, for a time sufficient for the cells or tissue to incorporate them; and c) optionally washing said sample of cells or tissue.
  • the solution comprising the photoactivatable compound (s) of the quinone family is the histological or cytological fixation solution.
  • Steps a) and b) are then implemented simultaneously.
  • the delay fixation of a cell suspension is very short (of the order of a few minutes) and that of tissue sample of the order of one hour.
  • the incorporation of photoactivatable compounds of the family of quinones, especially hypericin, by cells in suspension generally takes 1 to 2 hours.
  • the difference in the time of incorporation of the photoactivatable compounds of the family of quinones and in the time of fixation of the cells or tissues ensures that, according to the method of the invention, the photoactivatable compounds of the quinone family are incorporated while the cells have already been fixed, in other words are dead.
  • the photoactivatable compound (s) of the quinone family are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B. More preferably the method is a method for ex vivo cell labeling. or a tissue by hypericin.
  • the cells or the tissue are preferably tumor cells or suspected to be tumorous.
  • the sample of cells or tissue can thus be obtained by sampling or biopsy in a patient suspected of having a cancer and in whom it is sought to demonstrate the presence of tumor cells.
  • the sample may also have been obtained from a patient who has been diagnosed with cancer and for whom the efficacy of a therapeutic treatment is assessed by measuring the time course of hypericin incorporation by patients. cancer cells.
  • the cells or tissue can come from any organ, for example the uterus, liver, stomach, lung, esophagus, bladder, prostate, breast, etc.
  • a sample of cells may for example be a fluid sample such as urine, ascites, pleural or pericardial fluid; a smear such as cervical, vaginal, vulvar, or oesophageal smear; a malignant discharge or organ puncture, for example a superficial gland such as breast, thyroid and parotid or deep organ, such as the kidney, liver, ovaries, etc.
  • a tissue sample can be a biopsy or a total or partial excision of any organ.
  • the patient may be an animal, preferably a mammal such as a human, a rodent, a dog, a cat. Preferably the patient is a human.
  • the invention also relates to the use of a solution comprising a histological and cytological fixator and one or more photoactivatable compounds of the family of quinones, for the diagnosis of cancer or dysplasia, or the monitoring of the therapeutic efficacy of a treatment or cancer.
  • the invention more specifically proposes a method for diagnosing cancer or dysplasia, in which cells or tissue are stained with one or more photoactivatable compounds of the quinone family according to the method described above. and wherein the presence of cells or tissue labeled with the photoactivatable compound (s) of the quinone family is indicative of the presence of tumor or dysplastic cells.
  • the photoactivatable compound (s) of the quinone family are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B.
  • the method of diagnosing cancer or dysplasia comprising the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising hypericin for a time sufficient for the cells or tissue to incorporate hypericin; c) optionally washing said sample of cells or tissue; and d) detecting the presence of hypericin-labeled cells or tissue; wherein the presence of hypericin-labeled cells or tissue is indicative of the presence of cancer or dysplasia.
  • Detection of cell or tissue labeling by photoactivatable compounds of the quinone family can be easily performed by measuring the fluorescence emitted by the cells or the tissue, especially at around 600 nm for hypericin, hypocrellin A or hypocrelline B.
  • step d), and preferably all the steps are performed in an automated manner, thus allowing the automatic screening of samples.
  • perinuclear cell labeling is detected.
  • the invention is illustrated in more detail in the following examples, given without limitation.
  • Cells of a human strain glioblastoma are cultured on a petri dish.
  • Hypericin is suspended in Sakomano cytological fixative solution, with a concentration of 2 micromoles per ml, four hours before the experiments.
  • the fixative solution comprising hypericin is kept away from light.
  • the glioblastoma cells are fixed directly by placing them in the hypericin-based fixative solution for one hour.
  • Comparative tests are carried out with a microspectrofluorimeter to compare the level of fluorescence intensity and the quality of the fluorescence spectrum measured in cultures of identical live glioblastoma cells, incubated at the same concentration for one hour in Sakomano cytological fixative solution. containing hypericin, and the cells attached.
  • squamous cell carcinoma squamous cell carcinoma of the cervix type HELA are grown on a Petri dish. They are then fixed in a Sakomano-type cytological fixator already containing normal epithelial cells from a cervix smear in order to verify the affinity of hypericin for the tumor cells in a cell suspension containing several types. normal cells and tumor cells. Hypericin is then suspended for one hour in the fixed cell suspension at a concentration of 2 ⁇ M / ml and stored protected from light.
  • Tests are conducted with a microspectrofluorimeter to compare the level of fluorescence intensity and the quality of the fluorescence spectrum measured. for the different cells of the suspension, incubated at the same concentration for one hour in Sakomano cytological fixative solution containing rhypericin.
  • the cells thus having a particular marking can be detected by automatic recognition means. Also, it is possible to consider an automated screening of cytological samples including cervical smear in order to select only pathological cell smears.

Abstract

The invention relates to the combination, in a solution or in a kit, of a histological or cytological fixing solution and of one or more photoactivatable compounds of the family of quinones, in particular, hypericin, hypocrellin A and/or hyprcrellin B. The invention also relates to a method for labeling cells or tissues by one or more photoactivatable compounds of the family of quinones, in which the cells or tissues in contact with a histological or cytological fixing solution are labeled, simultaneously or subsequently, by the photoactivatable compound(s) of the family of quinones.

Description

COMBINAISON D ' UN FIXATEUR HISTOLOGIQUE OU CYTOLOGIQUE , ET D ' UN OU PLUSIEURS COMBINATION OF A HISTOLOGICAL OR CYTOLOGICAL FIXER AND ONE OR MORE
COMPOSES PHOTOACTIVABLES DE LA FAMILLE DES QUINONES , EN PARTICULIER L ' HYPERICINE , L ' HYPOCRELLINE A ET HYPOCRELLINE BPHOTOACTIVABLE COMPOUNDS OF THE FAMILY OF QUINONES, IN PARTICULAR HYPERICINE, HYPOCRELLIN A AND HYPOCRELLINE B
L'invention concerne la combinaison, dans une solution ou dans un kit, d'un fixateur histologique ou cytologique, et d'un ou plusieurs composés photoactivables de la famille des quinones.The invention relates to the combination, in a solution or kit, of a histological or cytological fixator, and one or more photoactivatable compounds of the family of quinones.
L'invention concerne également une méthode de marquage de cellules ou tissus par un ou plusieurs composés photoactivables de la famille des quinones, dans laquelle les cellules ou tissus mis en contact avec un fixateur histologique ou cytologique sont marqués, simultanément ou après, par le ou les composés photoactivables de la famille des quinones.The invention also relates to a method for labeling cells or tissues with one or more photoactivatable compounds of the quinone family, wherein the cells or tissues contacted with a histological or cytological fixator are labeled, simultaneously or afterwards, with the photoactivatable compounds of the family of quinones.
Des composés photoactivables de la famille des quinones incluent en particulier des quinones polycycliques telles que l'hypericine, l'hypocrelline A ou l'hypocrelline B, ou des dérivés de celles-ci.Photoactivatable compounds of the quinone family include, in particular, polycyclic quinones such as hypericin, hypocrelline A or hypocrelline B, or derivatives thereof.
"Dérivé" signifie un composé modifié chimiquement dans lequel la modification est considérée comme une routine par le chimiste à compétences ordinaires, tel qu'un ester ou un amide d'un acide, des groupes de protection, tels qu'un groupe benzyle pour un alcool ou un thiol, et un groupe tert-butoxycarbonyle pour une aminé."Derivative" means a chemically modified compound in which the modification is considered routine by the ordinary skill chemist, such as an ester or amide of an acid, protecting groups, such as a benzyl group for a alcohol or a thiol, and a tert-butoxycarbonyl group for an amine.
L'hypocrelline A (1 H-Cyclohepta[ghi]perylene-5,11-dione, 1-acétyl-2,3- dihydro-2,6,12-trihydroxy- 4,8,9,13-tétraméthoxy-2-méthyl-, cis-) et Phypocrelline BHypocrelline A (1H-Cyclohepta [ghi] perylene-5,11-dione, 1-acetyl-2,3-dihydro-2,6,12-trihydroxy-4,8,9,13-tetramethoxy-2- methyl-, cis-) and hypocrelline B
(1 H-Cyclohepta[ghi]perylene-5,12-dione, 3-acétyl-6,11-dihydroxy-4,8,9,13- tétramethoxy-2-méthyl) sont des quinones isolées à partir des champignons Hypocrella bambusae et Shiraia bambusicola. (1H-Cyclohepta [ghi] perylene-5,12-dione, 3-acetyl-6,11-dihydroxy-4,8,9,13-tetramethoxy-2-methyl) are quinones isolated from fungi Hypocrella bambusae and Shiraia bambusicola.
hypocrelline A hypocrelline Bhypocrellin A hypocrellin B
Ces pigments ont été utilisés en combinaison avec une photothérapie pour le traitement de différentes maladies de la peau. Récemment des propriétés antivirales ont été identifiées et il a été montré que ces quinones constituent de puissants photosensibilisants dans le traitement photodynamique du cancerThese pigments have been used in combination with phototherapy for the treatment of various skin diseases. Recently, antiviral properties have been identified and these quinones have been shown to be powerful photosensitizers in the photodynamic treatment of cancer.
(Hirayama et al., 1997 ; Zhang et al., 1998).(Hirayama et al., 1997, Zhang et al., 1998).
Les maxima d'absorption et d'émission de fluorescence, mesurés dans le méthanol, sont respectivement 463 nm et 600 nm pour l'hypocrelline A, et 459 nm etThe absorption and fluorescence emission maxima, measured in methanol, are respectively 463 nm and 600 nm for hypocrelline A, and 459 nm and
616 nm pour l'hyprocrelline B.616 nm for hyprocrellin B.
L'hypericine a pour formule chimique 1 , 3,4,6,8, 13-Hexahydroxy-10,11 -di- méthylphénanthro(1 ,10,9,8,opqra)perylène-7,14-dione :Hypericin has the chemical formula 1, 3,4,6,8,13-Hexahydroxy-10,11-dimethylphenanthro (1,10,9,8, opqra) perylene-7,14-dione:
L'hypericine est un pigment naturel isolé de plantes du genre Hypericum, qui constitue un puissant inhibiteur sélectif de la protéine kinase C.Hypericin is a natural pigment isolated from plants of the genus Hypericum, which is a potent selective inhibitor of protein kinase C.
L'hypericine présente une diversité de propriétés pharmacologiques qui vont d'une activité antibactérienne ou antivirale, à une activité antinéoplasique et à l'induction de l'apoptose. L'hypericine est connue de longue date pour ses effets photosensibilisants et comme entrant dans la composition d'un antidépresseur (le millepertuis).Hypericin has a variety of pharmacological properties ranging from antibacterial or antiviral activity to antineoplastic activity and induction of apoptosis. Hypericin has long been known for its effects photosensitizers and as part of the composition of an antidepressant (St. John's Wort).
C'est une molécule fluorescente qui présente un maximum d'absorption à 591 nm et un maximum d'émission de fluorescence à 594 nm, mesuré dans 5 l'éthanol.It is a fluorescent molecule that has an absorption maximum at 591 nm and a fluorescence emission maximum at 594 nm, measured in ethanol.
Il a été montré que l'hypericine perfusée dans la vessie s'accumule sélectivement dans les cellules carcinomateuses urothéliales. Cette caractéristique, associée à ses propriétés photosensibilisantes et fluorescentes, a conduit plusieurs équipes à proposer un diagnostic du cancer de la vessie par cytologie de 0 fluorescence ex vivo (Olivo et al., 2003a ; Olivo et al., 2003b ; Pytel et Schmeller, 2002). L'utilisation d'hypericine en tant que traitement photodynamique des lésions carcinomateuses de la vessie a été également proposée (Kamuhabwa et al., 2004). Ces méthodes impliquent l'infusion in vivo d'hypericine dans la vessie du patient.Hypericin infused into the bladder has been shown to accumulate selectively in urothelial carcinomatous cells. This characteristic, associated with its photosensitizing and fluorescent properties, led several teams to propose a diagnosis of bladder cancer by fluorescence cytology ex vivo (Olivo et al., 2003a, Olivo et al., 2003b, Pytel and Schmeller, 2002). The use of hypericin as a photodynamic treatment of carcinomatous lesions of the bladder has also been proposed (Kamuhabwa et al., 2004). These methods involve in vivo infusion of hypericin into the patient's bladder.
Il est connu que l'hypericine est une molécule lipophile qui peut être 5 incorporée dans la bicouche lipidique des membranes cellulaires. Les mécanismes exacts d'intégration ne sont pas connus mais il est supposé que cette intégration est médiée par un transport actif. Aussi, à ce jour l'hypericine n'a été utilisée que sur des cellules vivantes.It is known that hypericin is a lipophilic molecule that can be incorporated into the lipid bilayer of cell membranes. The exact integration mechanisms are not known but it is assumed that this integration is mediated by active transport. So far, hypericin has only been used on living cells.
Jusqu'à présent, il était en effet considéré comme nécessaire de pratiquer 0 un marquage à l'hypericine sur des cellules vivantes, par exemple en cultivant in vitro des cellules dans une solution contenant de l'hypericine, ou encore en mettant l'hypericine extemporanément en contact des cellules ou des tissus fraîchement prélevés, de manière à ce que l'hypericine puisse être incorporée par les cellules vivantes.Until now, it has indeed been considered necessary to practice hypericin labeling on living cells, for example by culturing in vitro cells in a solution containing hypericin, or by hypericin extemporaneously in contact with freshly harvested cells or tissues, so that hypericin can be incorporated by living cells.
-5 Contre toute attente, l'inventeur a maintenant démontré que l'hypericine, mélangée à un fixateur, est incorporée très rapidement par des cellules tumorales mises en suspension dans le mélange fixateur/hypericine, alors même que les cellules ont été tuées par le contact avec le fixateur. La mise en évidence de ce que l'hypericine est fixée aussi efficacement par les cellules, qu'elles soient vivantes ouAgainst all odds, the inventor has now demonstrated that hypericin, mixed with a fixative, is incorporated very rapidly by tumor cells suspended in the fixative / hypericin mixture, even though the cells have been killed by the contact with the fixer. The demonstration that hypericin is fixed as effectively by cells, whether they are alive or
O mortes, ouvre de nouvelles possibilités pour le marquage cytologique ou histologique à l'hypericine, et plus généralement à l'aide de composés photoactivables de la famille des quinones.O dead, opens new possibilities for cytological or histological staining with hypericin, and more generally with the help of photoactivatable compounds of the quinone family.
La fixation est une opération destinée à tuer les cellules pour les conserver, autant que possible, en l'état où elles se trouvaient pendant la vie. Faute de fixation, il y a risque d'autolyse. Les meilleurs fixateurs sont ceux qui, tout en agissant rapidement, produisent le moins possible de modifications secondaires ou d'artifices susceptibles de donner une idée très fausse de la morphologie interne des cellules. Désormais, il est possible d'étudier les cellules ou les tissus à tout moment, sans être obligé de les maintenir en vie, en réalisant un marquage avec un ou plusieurs composés photoactivables de la famille des quinones, notamment à l'hypericine, simultanément ou consécutivement à la fixation des cellules ou tissus par un fixateur cytologique ou histologique. En outre, il a été mis en évidence que lorsque des cellules tumorales sont mises en suspension dans une solution de fixateur contenant des doses micromolaires d'hypericine, l'affinité de l'hypericine pour les cellules est telle que la quasi-totalité de l'hypericine est incorporée par les cellules. La phase liquide de la suspension cellulaire ne contenant pratiquement plus d'hypericine, il est alors possible de réaliser une analyse directe des cellules (analyse morphologique et/ou de l'émission de fluorescence) sans rinçage de la suspension cellulaire. Cette propriété permet donc d'envisager une automatisation de la préparation cellulaire/tissulaire et de l'analyse des cellules.Fixation is an operation intended to kill the cells in order to preserve them, as much as possible, in the state they were during life. fault fixation, there is a risk of autolysis. The best fixators are those which, while acting rapidly, produce as little as possible of secondary modifications or artifices likely to give a very false idea of the internal morphology of the cells. Now, it is possible to study cells or tissues at any time, without having to keep them alive, by marking with one or more photoactivatable compounds of the quinone family, especially with hypericin, simultaneously or following the fixation of the cells or tissues by a cytological or histological fixator. Furthermore, it has been demonstrated that when tumor cells are suspended in a fixative solution containing micromolar doses of hypericin, the affinity of hypericin for cells is such that substantially all hypericin is incorporated by the cells. Since the liquid phase of the cell suspension contains practically no hypericin, it is then possible to carry out a direct analysis of the cells (morphological analysis and / or fluorescence emission) without rinsing the cell suspension. This property therefore makes it possible to envisage automation of the cell / tissue preparation and of the cell analysis.
L'invention concerne donc une solution comprenant un fixateur histologique ou cytologique et un ou plusieurs composés photoactivables de la famille des quinones. De préférence, le ou les composés photoactivables de la famille des quinones sont sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B. De manière préférentielle encore, ladite composition comprend, ou est constituée de, un fixateur histologique ou cytologique et d'hypericine.The invention therefore relates to a solution comprising a histological or cytological fixator and one or more photoactivatable compounds of the quinone family. Preferably, the photoactivatable compound (s) of the family of quinones are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B. More preferably, said composition comprises, or consists of, a histological or cytological fixator and hypericin.
Par « fixateur histologique ou cytologique » ou « solution de fixation histologique ou cytologique », on entend une solution couramment utilisée en analyse histologique ou cytologique pour réaliser la fixation de cellules ou tissus. Des exemples de fixateurs cytologiques incluent des fixateurs à base d'alcool éthylique, d'alcool méthylique, ou de polyéthylène glycol (PEG). Des exemples de fixateurs histologiques incluent des fixateurs à base de formaldéhyde, comme une solution de formol pur à 10% (le « formol pur » correspondant à la solution aqueuse de formaldéhyde à 40%, généralement vendue dans le commerce sous la dénomination de « formol »), une solution de formol - NaCI (constituée par exemple par mélange de 10 ml de formol pur, 0,9 g de NaCI et 90 ml d'eau), une solution de formol - acide acétique (par exemple 100 ml de formol pur, 50 ml d'acide acétique glacial et 850 ml d'eau), le liquide de Bouin alcoolique (ou Duboscq Brasil ; par exemple 250 ml de formol pur, 70 ml d'acide acétique glacial, 5 g d'acide picrique et 680 ml d'éthanol à 70°). D'autres exemples de fixateurs incluent le fixateur alcool - formol - acide acétique (20 ml de formol pur, 50 ml d'acide acétique glacial, 750 ml d'alcool éthylique absolu, et 180 ml d'eau), ou encore des fixateurs à base de chlorure de zinc (formol 10%, 10-50 g/l de chlorure de zinc, et acide acétique 5% ou NaCI 9 g/l, éventuellement alcool absolu 75 %). La solution selon l'invention comprend avantageusement 1 à 20 micromoles/ml, de préférence 1 à 15 micromoles/ml, de préférence 1 à 10 micromoles/ml, et de préférence encore 1 à 2 micromoles/ml de composés photoactivables de la famille des quinones.By "histological or cytological fixator" or "histological or cytological fixation solution" is meant a solution commonly used in histological or cytological analysis to achieve the fixation of cells or tissues. Examples of cytological fixatives include fixatives based on ethyl alcohol, methyl alcohol, or polyethylene glycol (PEG). Examples of histological fixatives include formaldehyde-based fixatives, such as a 10% pure formalin solution ("pure formalin" corresponding to the 40% aqueous formaldehyde solution, generally sold commercially as "formalin""), A formalin-NaCl solution (constituted for example by mixing 10 ml of pure formalin, 0.9 g of NaCl and 90 ml of water), a solution of formaldehyde - acetic acid (for example 100 ml of pure formalin, 50 ml of glacial acetic acid and 850 ml of water ), alcoholic liquor liquor (or Duboscq Brasil, for example 250 ml of pure formalin, 70 ml of glacial acetic acid, 5 g of picric acid and 680 ml of 70 ° ethanol). Other examples of fixatives include alcohol-formaldehyde-acetic acid fixative (20 ml of pure formalin, 50 ml of glacial acetic acid, 750 ml of absolute ethyl alcohol, and 180 ml of water), or fixatives. based on zinc chloride (10% formalin, 10-50 g / l zinc chloride, and 5% acetic acid or NaCl 9 g / l, optionally 75% absolute alcohol). The solution according to the invention advantageously comprises 1 to 20 micromoles / ml, preferably 1 to 15 micromoles / ml, preferably 1 to 10 micromoles / ml, and more preferably 1 to 2 micromoles / ml of photoactivatable compounds of the family of quinones.
L'invention concerne également un kit comprenant un fixateur histologique ou cytologique et un ou plusieurs composés photoactivables de la famille des quinones. De préférence, le ou les composés photoactivables de la famille des quinones sont sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B. De préférence encore, le kit selon l'invention comprend un fixateur histologique ou cytologique et de l'hypericine. Le kit peut optionnellement comprendre en outre un tampon de lavage approprié, une notice d'utilisation ou toute autre solution ou dispositif approprié.The invention also relates to a kit comprising a histological or cytological fixator and one or more photoactivatable compounds of the quinone family. Preferably, the photoactivatable compound (s) of the family of quinones are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B. More preferably, the kit according to the invention comprises a histological fixator or cytological and hypericin. The kit may optionally further comprise an appropriate wash buffer, a user's manual or any other suitable solution or device.
L'invention concerne aussi une méthode de marquage ex vivo de cellules ou d'un tissu par un ou plusieurs composés photoactivables de la famille des quinones. Ladite méthode comprend les étapes consistant à : a) mettre en contact un échantillon de cellules ou de tissu avec une solution de fixation histologique ou cytologique ; b) simultanément ou consécutivement, mettre en contact ledit échantillon de cellules ou de tissu avec une solution comprenant un ou plusieurs composés photoactivables de la familles des quinones, pendant un temps suffisant pour que les cellules ou le tissu les incorporent; et c) éventuellement laver ledit échantillon de cellules ou de tissu.The invention also relates to a method for ex vivo labeling of cells or tissue with one or more photoactivatable compounds of the quinone family. The method comprises the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising one or more photoactivatable compounds of the quinone family, for a time sufficient for the cells or tissue to incorporate them; and c) optionally washing said sample of cells or tissue.
De manière préférentielle, la solution comprenant le ou les composés photoactivables de la famille des quinones est la solution de fixation histologique ou cytologique. Les étapes a) et b) sont alors mises en œuvre simultanément. Le délai de fixation d'une suspension cellulaire est très court (de l'ordre de quelques minutes) et celui d'échantillon tissulaire de l'ordre d'une heure. Or l'incorporation de composés photoactivables de la familles des quinones, notamment de l'hypericine, par des cellules en suspension prend généralement de 1 à 2 heures. La différence dans le délai d'incorporation des composés photoactivables de la familles des quinones et dans le délai de fixation des cellules ou tissus garantit que, selon la méthode de l'invention, les composés photoactivables de la familles des quinones sont incorporés alors que les cellules ont déjà été fixées, autrement dit sont mortes.Preferably, the solution comprising the photoactivatable compound (s) of the quinone family is the histological or cytological fixation solution. Steps a) and b) are then implemented simultaneously. The delay fixation of a cell suspension is very short (of the order of a few minutes) and that of tissue sample of the order of one hour. However, the incorporation of photoactivatable compounds of the family of quinones, especially hypericin, by cells in suspension generally takes 1 to 2 hours. The difference in the time of incorporation of the photoactivatable compounds of the family of quinones and in the time of fixation of the cells or tissues ensures that, according to the method of the invention, the photoactivatable compounds of the quinone family are incorporated while the cells have already been fixed, in other words are dead.
De préférence, le ou les composés photoactivables de la famille des quinones sont sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B. De préférence encore la méthode est une méthode de marquage ex vivo de cellules ou d'un tissu par l'hypericine.Preferably, the photoactivatable compound (s) of the quinone family are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B. More preferably the method is a method for ex vivo cell labeling. or a tissue by hypericin.
Les cellules ou le tissu sont préférentiellement des cellules tumorales ou suspectées d'être tumorales. L'échantillon de cellules ou de tissu peut ainsi être obtenu par prélèvement ou biopsie chez un patient suspecté d'être atteint d'un cancer et chez qui on cherche à mettre en évidence la présence de cellules tumorales. L'échantillon peut également avoir été obtenu d'un patient chez qui on a diagnostiqué un cancer et pour lequel on évalue l'efficacité d'un traitement thérapeutique en mesurant l'évolution au cours du temps de l'incorporation d'hypericine par les cellules cancéreuses.The cells or the tissue are preferably tumor cells or suspected to be tumorous. The sample of cells or tissue can thus be obtained by sampling or biopsy in a patient suspected of having a cancer and in whom it is sought to demonstrate the presence of tumor cells. The sample may also have been obtained from a patient who has been diagnosed with cancer and for whom the efficacy of a therapeutic treatment is assessed by measuring the time course of hypericin incorporation by patients. cancer cells.
Les cellules ou tissu peuvent provenir de n'importe que l'organe, par exemple l'utérus, le foie, l'estomac, le poumon, l'œsophage, la vessie, la prostate, sein, etc. Un échantillon de cellules peut par exemple être un prélèvement de liquide tel que les urines, l'ascite, le liquide pleural ou péricardique ; d'un frottis tel qu'un frottis cervical, vaginal, vulvaire, ou oesophagien ; d'un écoulement mamellaire ou encore d'une ponction d'organe, par exemple une glande superficielle telle que le sein, la thyroïde et la parotide ou d'organe profond, tel que le rein, le foie, les ovaires, etc. Un échantillon de tissu peut être une biopsie ou une exérèse totale ou en partie de n'importe quel organe. Le patient peut être un animal, de préférence un mammifère tel qu'un humain, un rongeur, un chien, un chat. De préférence le patient est un humain.The cells or tissue can come from any organ, for example the uterus, liver, stomach, lung, esophagus, bladder, prostate, breast, etc. A sample of cells may for example be a fluid sample such as urine, ascites, pleural or pericardial fluid; a smear such as cervical, vaginal, vulvar, or oesophageal smear; a malignant discharge or organ puncture, for example a superficial gland such as breast, thyroid and parotid or deep organ, such as the kidney, liver, ovaries, etc. A tissue sample can be a biopsy or a total or partial excision of any organ. The patient may be an animal, preferably a mammal such as a human, a rodent, a dog, a cat. Preferably the patient is a human.
L'invention concerne également l'utilisation d'une solution comprenant un fixateur histologique et cytologique et un ou plusieurs composés photoactivables de la famille des quinones, pour le diagnostic d'un cancer ou d'une dysplasie, ou le suivi de l'efficacité thérapeutique d'un traitement ou d'un cancer.The invention also relates to the use of a solution comprising a histological and cytological fixator and one or more photoactivatable compounds of the family of quinones, for the diagnosis of cancer or dysplasia, or the monitoring of the therapeutic efficacy of a treatment or cancer.
L'invention propose plus spécifiquement une méthode de diagnostic d'un cancer ou d'une dysplasie, dans laquelle on réalise un marquage de cellules ou d'un tissu par un ou plusieurs composés photoactivables de la famille des quinones selon la méthode décrite plus haut, et où la présence de cellules ou d'un tissu marqué par le ou les composés photoactivables de la famille des quinones est révélatrice de la présence de cellules tumorales ou dysplasiques. De préférence, le ou les composés photoactivables de la famille des quinones sont sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B.The invention more specifically proposes a method for diagnosing cancer or dysplasia, in which cells or tissue are stained with one or more photoactivatable compounds of the quinone family according to the method described above. and wherein the presence of cells or tissue labeled with the photoactivatable compound (s) of the quinone family is indicative of the presence of tumor or dysplastic cells. Preferably, the photoactivatable compound (s) of the quinone family are selected from the group consisting of hypericin, hypocrellin A and hypocrellin B.
Selon un mode de réalisation, la méthode de diagnostic d'un cancer ou d'une dysplasie comprenant les étapes consistant à : a) mettre en contact un échantillon de cellules ou de tissu avec une solution de fixation histologique ou cytologique ; b) simultanément ou consécutivement, mettre en contact ledit échantillon de cellules ou de tissu avec une solution comprenant de l'hypericine, pendant un temps suffisant pour que les cellules ou le tissu incorporent l'hypericine ; c) éventuellement laver ledit échantillon de cellules ou de tissu ; et d) détecter la présence de cellules ou tissu marqué par l'hypericine ; dans laquelle la présence de cellules ou tissu marqué par l'hypericine est révélatrice de la présence d'un cancer ou d'une dysplasie.According to one embodiment, the method of diagnosing cancer or dysplasia comprising the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising hypericin for a time sufficient for the cells or tissue to incorporate hypericin; c) optionally washing said sample of cells or tissue; and d) detecting the presence of hypericin-labeled cells or tissue; wherein the presence of hypericin-labeled cells or tissue is indicative of the presence of cancer or dysplasia.
La détection d'un marquage des cellules ou tissu par des composés photoactivables de la famille des quinones peut être aisément effectuée en mesurant la fluorescence émise par les cellules ou le tissu, notamment aux alentours de 600 nm pour l'hypericine, l'hypocrelline A ou l'hypocrelline B.Detection of cell or tissue labeling by photoactivatable compounds of the quinone family can be easily performed by measuring the fluorescence emitted by the cells or the tissue, especially at around 600 nm for hypericin, hypocrellin A or hypocrelline B.
Les résultats montrent qu'il existe une grande affinité de l'hypericine pour certaines cellules tumorales avec une localisation préférentielle périnucléaire, alors que les cellules normales ne montrent qu'une très faible affinité avec une localisation périphérique au niveau de la membrane cellulaire. Aussi, selon un mode de réalisation de l'invention, au moins l'étape d), et de préférence toutes les étapes, sont réalisées de manière automatisée, permettant ainsi le criblage automatique d'échantillons.The results show that there is a high affinity of hypericin for certain tumor cells with a preferential perinuclear localization, whereas normal cells show only a very weak affinity with a peripheral localization at the level of the cell membrane. Also, according to one embodiment of the invention, at least step d), and preferably all the steps, are performed in an automated manner, thus allowing the automatic screening of samples.
De préférence, on détecte alors un marquage périnucléaire des cellules. L'invention est illustrée plus en détail dans es exemples suivants, donnés à titre non limitatif.Preferably, then perinuclear cell labeling is detected. The invention is illustrated in more detail in the following examples, given without limitation.
EXEMPLES EXEMPLE 1EXAMPLES EXAMPLE 1
Des cellules d'un glioblastome de souche humaine sont cultivées sur boîte de Pétri.Cells of a human strain glioblastoma are cultured on a petri dish.
De l'hypericine est mise en suspension dans une solution de fixateur cytologique Sakomano, avec une concentration de 2 micromoles par ml, quatre heures avant les expérimentations. La solution de fixateur comprenant l'hypericine est conservée à l'abri de la lumière.Hypericin is suspended in Sakomano cytological fixative solution, with a concentration of 2 micromoles per ml, four hours before the experiments. The fixative solution comprising hypericin is kept away from light.
Les cellules de glioblastome sont fixées directement en les mettant dans la solution de fixateur préparée à base d'hypericine pendant une heure.The glioblastoma cells are fixed directly by placing them in the hypericin-based fixative solution for one hour.
Des tests comparatifs sont menés avec un microspectrofluorimètre pour comparer le niveau d'intensité de fluorescence et la qualité du spectre de fluorescence mesurés dans des cultures de cellules de glioblastome vivantes identiques, incubées à la même concentration pendant une heure dans la solution de fixateur cytologique Sakomano contenant de l'hypericine, et les cellules fixées.Comparative tests are carried out with a microspectrofluorimeter to compare the level of fluorescence intensity and the quality of the fluorescence spectrum measured in cultures of identical live glioblastoma cells, incubated at the same concentration for one hour in Sakomano cytological fixative solution. containing hypericin, and the cells attached.
Les résultats montrent que les spectres sont totalement superposables et que les intensités de fluorescence sont identiques entre les cellules encore vivantes et les cellules fixées.The results show that the spectra are completely superimposable and that the fluorescence intensities are identical between the still alive cells and the fixed cells.
EXEMPLE 2EXAMPLE 2
Des cellules d'une lignée de carcinome malpighien épidermoïde du col de l'utérus de type HELA sont cultivées sur boîte de Pétri. Elles sont ensuite fixées dans un fixateur cytologique de type Sakomano renfermant déjà des cellules épithéliales normales provenant d'un frottis du col de l'utérus de façon à vérifier l'affinité de l'hypericine pour les cellules tumorales dans une suspension cellulaire contenant plusieurs types de cellules normales et des cellules tumorales. De l'hypericine est ensuite mise en suspension pendant une heure dans la suspension cellulaire fixée à une concentration de 2 //m/ml et conservée à l'abri de la lumière.Cells of squamous cell carcinoma squamous cell carcinoma of the cervix type HELA are grown on a Petri dish. They are then fixed in a Sakomano-type cytological fixator already containing normal epithelial cells from a cervix smear in order to verify the affinity of hypericin for the tumor cells in a cell suspension containing several types. normal cells and tumor cells. Hypericin is then suspended for one hour in the fixed cell suspension at a concentration of 2 μM / ml and stored protected from light.
Des tests sont menés avec un microspectrofluorimètre pour comparer le niveau d'intensité de fluorescence et la qualité du spectre de fluorescence mesurés pour les différentes cellules de la suspension, incubées à la même concentration pendant une heure dans la solution de fixateur cytologique Sakomano contenant de rhypericine.Tests are conducted with a microspectrofluorimeter to compare the level of fluorescence intensity and the quality of the fluorescence spectrum measured. for the different cells of the suspension, incubated at the same concentration for one hour in Sakomano cytological fixative solution containing rhypericin.
Les résultats montrent qu'il existe une grande affinité de rhypericine pour les cellules tumorales HELA avec une localisation préférentielle très particulière péri nucléaire, alors que les cellules normales ne montrent qu'une très faible affinité avec une localisation périphérique au niveau de la membrane cellulaire.The results show that there is a high affinity of rhypericin for HELA tumor cells with a very particular preferential peri-nuclear localization, whereas normal cells show only a very weak affinity with a peripheral localization at the level of the cell membrane.
Les cellules présentant ainsi un marquage particulier peuvent être détectées par des moyens de reconnaissance automatiques. Aussi, il est possible d'envisager un criblage automatisé des prélèvements cytologiques et notamment du frottis du col de l'utérus afin de ne sélectionner que les étalements cellulaires pathologiques. The cells thus having a particular marking can be detected by automatic recognition means. Also, it is possible to consider an automated screening of cytological samples including cervical smear in order to select only pathological cell smears.
REFERENCESREFERENCES
Hirayama J., et al. (1997) Photoinactivation of virus infectivity by hypocrellin A ; Photochem. Photobiol. 66, 697 Kamuhabwa A, Agostinis P, Ahmed B, Landuyt W, van Cleynenbreugel B, van Poppel H, de Witte P. (2004) Hypericin as a potential phototherapeutic agent in superficial transitional cell carcinoma of the bladder. Photochem Photobiol Sci. ; 3(8):772-80. Epub 2004 Apr 2.Hirayama J., et al. (1997) Photoinactivation of virus infectivity by hypocrellin A; Photochem. Photobiol. 66, 697 Kamuhabwa A, Agostinis P, Ahmed B, Landuyt W, Cleynenbreugel van B, van Poppel H, de Witte P. (2004) Hypericin as a potential phototherapeutic agent in superficial transitional cell carcinoma of the bladder. Photochem Photobiol Sci. ; 3 (8): 772-80. Epub 2004 Apr 2.
Olivo M, Lau W, Manivasager V, Bhuvaneswari R, Wei Z, Soo KC, Cheng C, Tan PH. (2003b) Novel photodynamic diagnosis of bladder cancer: ex vivo fluorescence cytology using hypericin. lnt J Oncol. ; 23(6):1501-4.Olivo M, Lau W, Manivasager V, Bhuvaneswari R, Wei Z, Soo KC, Cheng C, Tan PH. (2003b) Novel photodynamic diagnosis of cancer bladder: ex vivo fluorescence cytology using hypericin. J Oncol. ; 23 (6): 1501-4.
Olivo M, Lau W, Manivasager V, Tan PH, Soo KC, Cheng C. (2003a) Macro-microscopic fluorescence of human bladder cancer using hypericin fluorescence cystoscopy and laser confocal microscopy. lnt J Oncol. ; 23(4):983-90. Pytel A, Schmeller N. (2002) New aspect of photodynamic diagnosis of bladder tumors: fluorescence cytology. Urology. ; 59(2):216-9.Olivo M, Lau W, Manivasager V, Tan Ph, Soo KC, Cheng C. (2003a) Macro-microscopic fluorescence of human cancer bladder using hypericin fluorescence cystoscopy and laser confocal microscopy. J Oncol. ; 23 (4): 983-90. Pytel A, Schmeller N. (2002) New appearance of photodynamic diagnosis of bladder tumors: fluorescence cytology. Urology. ; 59 (2): 216-9.
Zhang J., et al.; (1998) Photodynamic effects of hypocrellin A on three human malignant cell lines by inducing apoptotic cell death J. Photochem. Photobiol. B. 43, 106 Zhang J., et al .; (1998) Photodynamic effects of hypocrellin A. on the human cell line by inducing apoptotic cell death J. Photochem. Photobiol. B. 43, 106

Claims

REVENDICATIONS
1. Solution pour le marquage de cellules ou de tissus comprenant un fixateur histologique ou cytologique et un ou plusieurs composés photoactivables de la famille des quinones sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B.A solution for labeling cells or tissues comprising a histological or cytological fixator and one or more photoactivatable compounds of the family of quinones selected from the group consisting of hypericin, hypocrellin A and hypocrellin B.
2. Solution selon la revendication 1 , comprenant 1 à 20 micromoles /ml de composés photoactivables de la famille des quinones.2. Solution according to claim 1, comprising 1 to 20 micromoles / ml of photoactivatable compounds of the quinone family.
3. Solution selon l'une quelconque des revendications précédentes, comprenant un fixateur histologique ou cytologique et de l'hypericine.3. Solution according to any one of the preceding claims, comprising a histological or cytological fixator and hypericin.
4. Kit comprenant un fixateur histologique ou cytologique et un ou plusieurs composés photoactivables de la famille des quinones sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B.4. A kit comprising a histological or cytological fixator and one or more photoactivatable compounds of the quinone family selected from the group consisting of hypericin, hypocrellin A and hypocrellin B.
5. Kit selon Ia revendication 4, comprenant un fixateur histologique ou cytologique et de l'hypericine.5. Kit according to claim 4, comprising a histological or cytological fixator and hypericin.
6. Méthode de marquage ex vivo de cellules ou d'un tissu par un ou plusieurs composés photoactivables de la famille des quinones, ladite méthode comprenant les étapes consistant à : a) mettre en contact un échantillon de cellules ou de tissu avec une solution de fixation histologique ou cytologique ; b) simultanément ou consécutivement, mettre en contact ledit échantillon de cellules ou de tissu avec une solution comprenant un ou plusieurs composés photoactivables de la famille des quinones sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B, pendant un temps suffisant pour que les cellules ou le tissu incorporent le ou les composés photoactivables de la famille des quinones; c) éventuellement laver ledit échantillon de cellules ou de tissu. A method of ex vivo labeling of cells or tissue with one or more photoactivatable compounds of the quinone family, said method comprising the steps of: a) contacting a sample of cells or tissue with a solution of histological or cytological fixation; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising one or more photoactivatable compounds of the quinone family selected from the group consisting of hypericin, hypocrellin A and hypocrelline B, for a time sufficient for the cells or tissue to incorporate the photoactivatable compound (s) of the quinone family; c) optionally washing said sample of cells or tissue.
7. Méthode selon la revendication 6, dans laquelle ladite solution comprenant un ou plusieurs composés photoactivables de la famille des quinones est la solution de fixation histologique ou cytologique et les étapes a) et b) sont mises en œuvre simultanément.The method of claim 6, wherein said solution comprising one or more photoactivatable compounds of the quinone family is the histological or cytological fixation solution and steps a) and b) are carried out simultaneously.
8. Méthode selon la revendication 6 ou 7, dans laquelle on marque des cellules ou un tissu par l'hypericine.The method of claim 6 or 7, wherein cells or tissue are labeled with hypericin.
9. Méthode selon l'une quelconque des revendications 6 à 8, dans laquelle l'échantillon de cellules ou de tissu provient d'un patient suspecté d'être atteint d'un cancer.The method of any one of claims 6 to 8, wherein the cell or tissue sample is from a patient suspected of having cancer.
10. Méthode selon l'une quelconque des revendications 6 à 9, dans laquelle l'échantillon des cellules est un prélèvement de liquide, un frottis, un écoulement mamellaire, ou une ponction d'organe.The method of any one of claims 6 to 9, wherein the sample of the cells is a fluid sample, a smear, a mamellar flow, or an organ puncture.
11. Méthode selon l'une quelconque des revendications 6 à 10, dans laquelle l'échantillon de tissu est une biopsie ou une exérèse totale ou partielle d'un organe.The method of any one of claims 6 to 10, wherein the tissue sample is a biopsy or total or partial excision of an organ.
12. Utilisation d'une solution selon l'une quelconque des revendications 1 à 3, pour le diagnostic in vitro d'un cancer ou d'une dysplasie, ou pour le suivi de l'efficacité thérapeutique d'un traitement du cancer.12. Use of a solution according to any one of claims 1 to 3, for the in vitro diagnosis of cancer or dysplasia, or for monitoring the therapeutic efficacy of a cancer treatment.
13. Méthode de diagnostic in vitro d'un cancer ou d'une dysplasie comprenant les étapes consistant à : a) mettre en contact un échantillon de cellules ou de tissu avec une solution de fixation histologique ou cytologique ; b) simultanément ou consécutivement, mettre en contact ledit échantillon de cellules ou de tissu avec une solution comprenant un ou plusieurs composés photoactivables de la famille des quinones sélectionnés dans le groupe constitué de l'hypericine, de l'hypocrelline A et de l'hypocrelline B, pendant un temps suffisant pour que les cellules ou le tissu incorporent le ou les composés photoactivables de la famille des quinones ; c) éventuellement laver ledit échantillon de cellules ou de tissu ; et d) détecter la présence de cellules ou tissu marqué par le ou les composés photoactivables de la famille des quinones ; dans laquelle la présence de cellules ou tissu marqué par le ou les composés photoactivables de la famille des quinones est révélatrice de la présence d'un cancer ou d'une dysplasie.A method of in vitro diagnosis of cancer or dysplasia comprising the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising one or more photoactivatable compounds of the quinone family selected from the group consisting of hypericin, hypocrellin A and hypocrelline B, for a time sufficient for the cells or tissue to incorporate the photoactivatable compound (s) of the quinone family; c) optionally washing said sample of cells or tissue; and d) detecting the presence of cells or tissue labeled with the one or more photoactivatable compounds of the quinone family; wherein the presence of cells or tissue labeled with the one or more photoactivatable compounds of the quinone family is indicative of the presence of cancer or dysplasia.
14. Méthode de diagnostic in vitro d'un cancer ou d'une dysplasie selon la revendication 13, comprenant les étapes consistant à : a) mettre en contact un échantillon de cellules ou de tissu avec une solution de fixation histologique ou cytologique ; b) simultanément ou consécutivement, mettre en contact ledit échantillon de cellules ou de tissu avec une solution comprenant de l'hypericine, pendant un temps suffisant pour que les cellules ou le tissu incorporent l'hypericine ; c) éventuellement laver ledit échantillon de cellules ou de tissu ; et d) détecter la présence de cellules ou tissu marqué par l'hypericine ; dans laquelle la présence de cellules ou tissu marqué par l'hypericine est révélatrice de la présence d'un cancer ou d'une dysplasie.14. The in vitro diagnostic method of cancer or dysplasia according to claim 13, comprising the steps of: a) contacting a sample of cells or tissue with a histological or cytological fixation solution; b) simultaneously or consecutively, contacting said sample of cells or tissue with a solution comprising hypericin for a time sufficient for the cells or tissue to incorporate hypericin; c) optionally washing said sample of cells or tissue; and d) detecting the presence of hypericin-labeled cells or tissue; wherein the presence of hypericin-labeled cells or tissue is indicative of the presence of cancer or dysplasia.
15. Méthode selon la revendication 13 ou 14, dans laquelle au moins l'étape d) est réalisée de manière automatisée.15. The method of claim 13 or 14, wherein at least step d) is performed in an automated manner.
16. Méthode selon l'une des revendications 13 à 15, dans laquelle on détecte un marquage périnucléaire. 16. Method according to one of claims 13 to 15, wherein a perinuclear marking is detected.
EP06743663A 2005-04-07 2006-04-07 Combination consisting of a histological or cytological fixing solution and of one or more photoactivatable compounds of the family of quinones, in particular, hypericin, hypocrellin a and hyprcrellin b Withdrawn EP1865974A1 (en)

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FR0503487A FR2884144B1 (en) 2005-04-07 2005-04-07 COMBINATION OF A HISTOLOGICAL OR CYTOLOGICAL FIXER, AND ONE OR MORE PHOTOACTIVABLE COMPOUNDS OF THE FAMILY OF QUINONS, ESPECIALLY HYPERICINE, HYPOCRELLIN A AND HYPOCRELLINE B.
PCT/FR2006/000782 WO2006106245A1 (en) 2005-04-07 2006-04-07 Combination consisting of a histological or cytological fixing solution and of one or more photoactivatable compounds of the family of quinones, in particular, hypericin, hypocrellin a and hyprcrellin b

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