EP1865974A1 - Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l 'hypericine, l'hypocrelline a et hypocrelline b - Google Patents

Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l 'hypericine, l'hypocrelline a et hypocrelline 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
Authority
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.)
Withdrawn
Application number
EP06743663A
Other languages
German (de)
English (en)
French (fr)
Inventor
Peltier Eric
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maclip SARL
Original Assignee
Novacyt SA
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Novacyt SA filed Critical Novacyt SA
Publication of EP1865974A1 publication Critical patent/EP1865974A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP06743663A 2005-04-07 2006-04-07 Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l 'hypericine, l'hypocrelline a et hypocrelline b Withdrawn EP1865974A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0503487A FR2884144B1 (fr) 2005-04-07 2005-04-07 Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l'hypericine, l'hypocrelline a et l'hypocrelline b.
PCT/FR2006/000782 WO2006106245A1 (fr) 2005-04-07 2006-04-07 Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l 'hypericine, l'hypocrelline a et hypocrelline b

Publications (1)

Publication Number Publication Date
EP1865974A1 true EP1865974A1 (fr) 2007-12-19

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP06743663A Withdrawn EP1865974A1 (fr) 2005-04-07 2006-04-07 Combinaison d'un fixateur histologique ou cytologique, et d'un ou plusieurs composes photoactivables de la famille des quinones, en particulier l 'hypericine, l'hypocrelline a et hypocrelline b

Country Status (12)

Country Link
US (1) US20090047704A1 (zh)
EP (1) EP1865974A1 (zh)
JP (1) JP2008534975A (zh)
KR (1) KR20070116981A (zh)
CN (1) CN101180067A (zh)
AU (1) AU2006231137A1 (zh)
BR (1) BRPI0612367A2 (zh)
CA (1) CA2603662A1 (zh)
FR (1) FR2884144B1 (zh)
IL (1) IL186391A0 (zh)
RU (1) RU2007141205A (zh)
WO (1) WO2006106245A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090226059A1 (en) * 2008-02-12 2009-09-10 Richard Levenson Tissue Processing And Assessment
US20150104394A1 (en) * 2012-05-18 2015-04-16 Igr-Institut Gustave Roussy Characterization of biological tissues at a cellular level using red and far-red fluorescent dyes
GB2576331B8 (en) * 2018-08-14 2021-04-07 Apacor Ltd Fixative solution and method of preparation of biological sample for examination

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981163A (en) * 1990-05-15 1999-11-09 New York Blood Center, Inc. Process for the sterilization of biological compositions using irradiation and quenchers of type I and type II photodynamic reactions
AU3474593A (en) * 1992-01-16 1993-08-03 New York University Hypericin treatment of vaccine agents for improved immunogenicity
ES2159270B1 (es) * 2000-03-06 2002-04-01 Asac Compania De Biotecnologia Oleorresina de hypericum perforatum l., procedimiento de obtencion y usos.
IL143318A0 (en) * 2001-05-23 2002-04-21 Herbal Synthesis Corp Herbal compositions for the treatment of mucosal lesions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006106245A1 *

Also Published As

Publication number Publication date
CA2603662A1 (fr) 2006-10-12
US20090047704A1 (en) 2009-02-19
RU2007141205A (ru) 2009-05-20
FR2884144A1 (fr) 2006-10-13
FR2884144B1 (fr) 2008-04-11
BRPI0612367A2 (pt) 2010-11-03
KR20070116981A (ko) 2007-12-11
WO2006106245A1 (fr) 2006-10-12
IL186391A0 (en) 2008-01-20
JP2008534975A (ja) 2008-08-28
CN101180067A (zh) 2008-05-14
AU2006231137A1 (en) 2006-10-12

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