WO2006053680A1 - Procede permettant de diagnostiquer un cancer ex vivo - Google Patents

Procede permettant de diagnostiquer un cancer ex vivo Download PDF

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Publication number
WO2006053680A1
WO2006053680A1 PCT/EP2005/012083 EP2005012083W WO2006053680A1 WO 2006053680 A1 WO2006053680 A1 WO 2006053680A1 EP 2005012083 W EP2005012083 W EP 2005012083W WO 2006053680 A1 WO2006053680 A1 WO 2006053680A1
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WO
WIPO (PCT)
Prior art keywords
alkaloid
cancer
sample
compound
derivative
Prior art date
Application number
PCT/EP2005/012083
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English (en)
Inventor
Wassyl Nowicky
Original Assignee
Nowicky Wassili
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 Nowicky Wassili filed Critical Nowicky Wassili
Publication of WO2006053680A1 publication Critical patent/WO2006053680A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • the present invention is in the field of medical diagnostics and relates to an in vitro method of detection of a pathological condition, particularly of a malignant tumor, in a human individual.
  • blood cells particularly white blood cells
  • blood cells obtained from cancer patients differ significantly over their counterparts obtained from healthy blood donors as regards their physiological status and/or metabolic activity, for instance their ability to take up nutritional compounds such as, e.g. amino acids, in the presence of alkaloid derivatives, particularly in the presence of one or more alkaloid derivatives having a quaternary nitrogen atom such as the ones present in the anti-cancer drug Ukrain®, in vitro.
  • This ex vivo method is extremely patient-friendly as it does not require administration of any possibly hazardous agent to a patient solely for the purpose of cancer diagnostics.
  • the present invention therefore in its first aspect relates to a method of detecting a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or a cancer disease, in an individual, the method comprising:
  • the invention relates to such a method, wherein said liquid sample is selected from the group consisting of a heparinised whole blood sample, a saliva sample, and a sweat sample.
  • the invention relates to such a method, wherein said alkaloid component comprises at least one alkaloid, preferably a mixture of alkaloids, present in the herb Chelidonium majus L.
  • the invention relates to such a method, wherein said alkaloid component comprises at least one quaternary alkaloid or alkaloid derivative.
  • the invention relates to such a method, wherein said quaternary alkaloid or alkaloid derivative is present in or derivable from the herb Chelidonium majus L. by chemical modification, particularly by alkylation, and preferably comprises a quaternary chelidonine or chelidonine derivative.
  • the invention relates to such a method, wherein said alkaloid component comprises a mixture of compounds obtained by alkylation of some or all alkaloids present in the herb Chelidonium majus L., and most preferably comprises the anti-cancer composition Ukrain®.
  • the invention relates to such a method, wherein the sample is subjected to incubation at 37 0 C for a period of up to 4 hours.
  • the invention relates to such a method, wherein said alkaloid component is added at a concentration of from 0.1 micrograms active ingredient per 1 ml of said liquid sample to 0.5 milligrams active ingredient per 1 ml of said liquid sample.
  • the invention relates to such a method, wherein said analytical determination comprises fluorescence detection of single compounds present in said cell-free fraction, or of said fraction as a whole.
  • the invention relates to such a method, comprising separating said cell-free fraction into single compound fractions by HPLC and obtaining a concentration profile of at least one, preferably of a plurality of compound fractions, by fluorescence detection.
  • the invention relates to such a method, comprising subjecting said cell-free fraction as a whole to fluorescence detection over a range of emission wavelengths.
  • the invention relates to such a method, comprising subjecting said cell-free fraction to at least two different excitation wavelengths, preferably to excitation wavelengths of 350 nm or 410 nm.
  • the invention relates to such a method, comprising adding to said liquid sample at least one traceable nutritional compound, and optionally a suitable cell culture medium, and measuring an uptake of said at least one traceable nutritional compound by the cells, preferably through detection of a corresponding concentration decline in the cell-free fraction, wherein an increased uptake of said traceable nutritional compound by the cells of said individual as compared to the controls indicates a pathological condition, particularly the presence of a malignancy, more particularly of a malignant tumor or a cancer disease.
  • the invention relates to such a method, wherein to the liquid samples a suitable cell culture medium is added and wherein said cell culture medium is RPMI 1 640 supplemented with L-glutamine.
  • the invention relates to such a method, wherein the traceable nutritional compound is a radioisotope-labeled compound, preferably a radioisotope-labeled amino acid, particularly tritium-labeled L- leucine.
  • the traceable nutritional compound is a radioisotope-labeled compound, preferably a radioisotope-labeled amino acid, particularly tritium-labeled L- leucine.
  • the invention relates to such a method, wherein the radioisotope-labeled compound is added at a concentration of 1 to 5, preferably 2 to 3 ⁇ Ci per 1 ml of cell culture medium.
  • the invention relates to such a method, wherein incubation is carried out at a temperature of 37° C and under a 5% CO 2 atmosphere.
  • the invention relates to such a method, wherein the alkaloid component comprises a quaternary alkaloid derivative which comprises as a fourth ligand to the quaternary nitrogen a residue selected from the group consisting of a -H, -OH, -SH, aliphatic, alkyl, aryl, aralkyl, alkyloxy, aryloxy, and a halogen residue.
  • the alkaloid component comprises a quaternary alkaloid derivative which comprises as a fourth ligand to the quaternary nitrogen a residue selected from the group consisting of a -H, -OH, -SH, aliphatic, alkyl, aryl, aralkyl, alkyloxy, aryloxy, and a halogen residue.
  • the invention relates to an alkaloid component, preferably comprising a quaternary alkaloid derivative, for use as an ex vivo diagnostic tool in the detection of malignancy in a mammal, particularly of a malignant tumor or cancer in an animal or human individual.
  • the invention further relates to such an alkaloid component, which comprises an alkaloid derivative that adheres to or accumulates in cancer cells or tissues, the alkaloid derivative preferably being derived from an alkaloid present in the herb Chelidonium majus L.
  • the invention further relates to such an alkaloid component, wherein the alkaloid component comprises a mixture of compounds obtained by alkylation of some or all alkaloids present in the herb Chelidonium majus L., and most preferably comprises the anti-cancer composition Ukrain®.
  • the invention further relates to such an alkaloid component, which comprises at least one quaternary alkaloid or alkaloid derivative, particularly at least one quaternary alkaloid or alkaloid derivative that adheres to or accumulates in cancer cells or tissues, and preferably comprises a quaternary chelidonine or chelidonine derivative.
  • the invention relates to the use of such an alkaloid component, preferably comprising a quaternary alkaloid derivative, for the manufacture of an ex vivo diagnostic tool, particularly for the manufacture of a diagnostic kit, for the detection of malignancy in a mammal, particularly of a malignant tumor or cancer in an animal or human individual.
  • an alkaloid component preferably comprising a quaternary alkaloid derivative
  • Example 1 Uptake of radioactivity by blood cells upon exposure to
  • heparinised blood obtained from a colon cancer patient were added per well of a 96-well microtiter plate containing the cell culture medium RPMI-1640 supplemented with L-glutamine (300 mg/L). Additionally, 100 ⁇ L of the commercially available drug Ukrain (Nowicky Pharma, Austria; ampoules containing 5 mg active ingredient per 5ml aqueous solution; Ukrain® is a registered trademark of Nowicky Pharma AG) were added to the cell suspension in the experimental group, while the controls were left without an addition of Ukrain. The cell suspensions were subjected to incubation at 37 0 C under 5% CO2 atmosphere for a period of 4 hours.
  • the observed effect is mainly if not exclusively inferred by the white blood cells, e.g. leukocytes.
  • the exciting results demonstrate that the addition of Ukrain to blood cell cultures of diseased subjects, particularly of cancer patients, causes physiological effects to white and/or red blood cells in vitro resulting in an increased uptake of nutritional compounds, particularly of precursors to the protein synthesis, i.e. amino acids, thus providing an important information as to the presence of malignancy in a said subject.
  • Example 1 was performed using radioisotope- labeled L-Ieucine as a traceable nutritional compound for uptake by the blood cells it is understood by those of ordinary skill in the art that other traceable nutritional compounds, such as fluorescent or fluorescence-labeled compounds or other compounds that may be visualized by imaging methods, may be used in alternative and will thus be within the scope of the present claims without deviating from the spirit of the invention.
  • Example 2 Blood samples of diseased and healthy donors incubated in the presence of Ukrain
  • 5ml venous blood were taken from a patient and transferred to a heparinized glass vial. 1 ml thereof was transferred into an Eppendorf microcentrifuge glass container and mixed with 100 microliter of an Ukrain solution at varying dilution rates, i.e. comprising Ukrain dissolved in redistilled water at volumetric ratios of 1 :5, 1 : 10, 1 : 100, or 1 : 1000.
  • homotaurine (Sigma Aldrich) was used as an internal standard.
  • the protein- free extracts obtained by this procedure were stored at -20°C and kept for subsequent analysis.
  • Various compounds including a number of amino acids were determined qualitatively and quantitatively by HPLC (Agilent 1 100), applying isocratic elution from a diasorb 130 C16 T, 6 micrometer, 3x250 nm column (Elsiko, Russia); mobile phase: 0.1 M sodium acetate buffer, ph 5.71 - acetonitryl: water (1 :1 ) 65:35; flow rate 0.6 ml/min, 3O 0 C.
  • a commercially available amino acid mixture (Fluka) was applied. The statistical evaluation was done using the software Statistica 6.0.
  • Example 3 Fluorescence detection in plasma samples - comparison of emission intensity at different wavelengths
  • Plasma samples have been prepared and exposed to Ukrain concentrations according to Example 2.
  • the plasma fractions obtained therefrom have been subjected to fluorescence detection using different excitation wavelengths, i.e. 350 nm or 410 nm.
  • the resulting fluorescence spectra revealed surprising and most interesting differences with samples taken from cancer patients as opposed to those taken from healthy donors.
  • the emission intensities at 450 nm (excitation wavelength 350 nm) and particularly at 520 nm (excitation wavelength 410 nm) of plasma from Ukrain-treated blood samples of diseased patients significantly exceeded the controls of healthy donors, as can be seen in Figures 3 and 4.
  • the addition of Ukrain to the blood samples causes an increase in fluorescence emission of up to four times the value of the Ukrain-free samples, which increase is significantly higher with samples from cancer patients than from healthy donors.
  • Example 4 Fluorescence detection in other bodily fluids - comparison of emission intensity at different wavelengths Example 3 was repeated except that the blood samples were replaced by samples of other bodily fluids, particularly by sweat and saliva samples. The samples were treated like the blood samples described in Examples 2 and 3 and subjected to fluorescence detection as described there.
  • results were very similar to those obtained with blood samples, thus confirming the diagnostic value of this method for the detection of a malignancy, particularly of the presence of tumor cells, solid or non-solid tumors including metastases, or cancer in mammals and particularly in human individuals.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention se rapporte à un procédé de diagnostic ex vivo permettant de détecter un développement tumorigène malin ou un cancer chez un sujet humain, ledit procédé étant fondé sur la détermination différentielle d'au moins un composé présent dans un fluide corporel d'un donneur malade ou d'un donneur sain avant et après l'addition d'un composé alcaloïde, particulièrement du mélange disponible dans le commerce de dérivés alcaloïdes connus sous le nom de UKRAIN®. L'invention se rapporte également à une trousse de diagnostic permettant la mise en oeuvre de ce procédé ainsi qu'à l'utilisation de ces dérivés alcaloïdes en tant qu'outils diagnostiques pour diagnostiquer un cancer.
PCT/EP2005/012083 2004-11-19 2005-11-11 Procede permettant de diagnostiquer un cancer ex vivo WO2006053680A1 (fr)

Applications Claiming Priority (2)

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EP04027575.2 2004-11-19
EP04027575 2004-11-19

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007107334A1 (fr) * 2006-03-20 2007-09-27 Nowicky Wassili Procédé de diagnostic du cancer ex vivo
WO2007073919A3 (fr) * 2005-12-28 2007-11-22 Nowicky Wassili Procede de detection du cancer
WO2012086802A1 (fr) * 2010-12-24 2012-06-28 アークレイ株式会社 Procédé de détection d'une cellule cancéreuse

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2110533A (en) * 1981-07-13 1983-06-22 Nowicky Wassili Compositions containing alkaloid derivatives
US4816462A (en) * 1982-05-18 1989-03-28 Nowicky Wassili Method for diagnosing and for the therapeutic treatment of tumors and/or infectious diseases of different types with alkaloid-compounds
US5399338A (en) * 1991-05-01 1995-03-21 University Of New Mexico Enhancement of abnormal tissue uptake of antibodies, tumor-specific agents or conjugates thereof for diagnostic imaging or therapy
US5567593A (en) * 1992-07-28 1996-10-22 Beljanski; Mirko Cytodiagnostic method using alstonine as a selective marker, and diagnostic kit containing marker
WO2000006143A1 (fr) * 1998-07-27 2000-02-10 Texas Pharmaceuticals, Inc. Hyperthermie intracellulaire induite chimiquement
WO2003041721A1 (fr) * 2001-11-15 2003-05-22 Nowicky Wassili Processus de reaction d'alcaloides et utilisation des produits de reaction dans la preparation de medicaments

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2110533A (en) * 1981-07-13 1983-06-22 Nowicky Wassili Compositions containing alkaloid derivatives
US4816462A (en) * 1982-05-18 1989-03-28 Nowicky Wassili Method for diagnosing and for the therapeutic treatment of tumors and/or infectious diseases of different types with alkaloid-compounds
US5399338A (en) * 1991-05-01 1995-03-21 University Of New Mexico Enhancement of abnormal tissue uptake of antibodies, tumor-specific agents or conjugates thereof for diagnostic imaging or therapy
US5567593A (en) * 1992-07-28 1996-10-22 Beljanski; Mirko Cytodiagnostic method using alstonine as a selective marker, and diagnostic kit containing marker
WO2000006143A1 (fr) * 1998-07-27 2000-02-10 Texas Pharmaceuticals, Inc. Hyperthermie intracellulaire induite chimiquement
WO2003041721A1 (fr) * 2001-11-15 2003-05-22 Nowicky Wassili Processus de reaction d'alcaloides et utilisation des produits de reaction dans la preparation de medicaments

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DOROSHENKO Y M ET AL: "A method for determination of Ukrain in blood plasma for monitoring and pharmacokinetic study", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, vol. 26, no. 5-6, 2000, pages 163 - 170, XP008060482, ISSN: 0378-6501 *
DOROSHENKO Y M ET AL: "The dynamics of concentration of the main fluorescent component of Ukrain in the tissues and blood plasma of rats with W-256 tumor after a single intravenous injection", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, vol. 26, no. 5-6, 2000, pages 171 - 177, XP008060378, ISSN: 0378-6501 *
DUNN B P ET AL: "EFFECTS OF THE ANTI NEOPLASTIC ALKALOID ACRONYCINE ON NUCLEOSIDE UPTAKE AND INCORPORATION INTO NUCLEIC-ACIDS BY CULTURED L-5178-Y CELLS", CANCER RESEARCH, vol. 33, no. 10, 1973, pages 2310 - 2319, XP008060587, ISSN: 0008-5472 *
IOANNONI B ET AL: "TETRANDRINE AND TRANSMEMBRANE SIGNAL TRANSDUCTION EFFECT OF PHOSPHOINOSITIDE METABOLISM CALCIUM FLUX AND PROTEIN KINASE C TRANSLOCATION IN HUMAN LYMPHOCYTES", INTERNATIONAL ARCHIVES OF ALLERGY AND APPLIED IMMUNOLOGY, vol. 89, no. 4, 1989, pages 349 - 354, XP008060588, ISSN: 0020-5915 *
JAGIELLO-WOJTOWICZ E ET AL: "Preliminary pharmacokinetic studies of Ukrain in rats", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, vol. 24, no. 5-6, 1998, pages 309 - 311, XP008060484, ISSN: 0378-6501 *
KENNEDY B: "NUCLEOSIDE UPTAKE ACRONYCINE ACTION AND MEMBRANE FLUIDITY DURING LYMPHOCYTE ACTIVATION", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 7, no. 5, 1979, & 53RD MEETING OF THE BIOCHEMICAL SOCIETY, CAMBRIDGE, ENGLAND, JUNE 27-29, 1979. BIOCHEM SOC TRANS., pages 1002 - 1003, XP008060586, ISSN: 0300-5127 *
NEFYODOV L I ET AL: "Amino acids and their derivatives in blood plasma of patients with breast cancer treated with Ukrain. Part V", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH, vol. 22, no. 3-5, 1996, pages 155 - 157, XP008060480, ISSN: 0378-6501 *
NOWICKY J W ET AL: "MACROSCOPIC UV-MARKING THROUGH AFFINITY", JOURNAL OF TUMOR MARKER ONCOLOGY, vol. 3, no. 4, 1988, pages 463 - 466, XP008060373, ISSN: 0886-3849 *
OSTER J B ET AL: "ENHANCEMENT OF CELLULAR UPTAKE OF ELLIPTICINE BY INSULIN PRE INCUBATION", EUROPEAN JOURNAL OF CANCER AND CLINICAL ONCOLOGY, vol. 17, no. 10, 1981, pages 1097 - 1104, XP008060589, ISSN: 0277-5379 *
SLESAK B ET AL: "In vitro effects of Chelidonium majus L. alkaloid thiophosphoric acid conjugates (Ukrain) on the phenotype of normal human lymphocytes.", DRUGS UNDER EXPERIMENTAL AND CLINICAL RESEARCH. 1992, vol. 18 Suppl, 1992, pages 17 - 21, XP008060487, ISSN: 0378-6501 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007073919A3 (fr) * 2005-12-28 2007-11-22 Nowicky Wassili Procede de detection du cancer
WO2007107334A1 (fr) * 2006-03-20 2007-09-27 Nowicky Wassili Procédé de diagnostic du cancer ex vivo
WO2012086802A1 (fr) * 2010-12-24 2012-06-28 アークレイ株式会社 Procédé de détection d'une cellule cancéreuse

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TW200632319A (en) 2006-09-16
AR051502A1 (es) 2007-01-17

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