Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-02-22
1996-10-22
Weimar, Elizabeth C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 721, 435 39, 435808, 435810, 435968, 436 63, 436 64, 436164, 436172, 436805, 436813, 436816, 514960, 514962, 424408, G01N 33574, G01N 3348, A01N 2534
Patent
active
055675930
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to cytodiagnostic techniques. More particularly, it relates to the detection of tumors, notably for the establishment of a tumoral diagnosis, and to the detection of chromosomal aberrations by means of alstonine, using a procedure of selective detection by fluorescence.
In the present description, the term "cytodiagnostic" is understood to include both the diagnosis of tumoral diseases and cytogenetics.
Alstonine is an alkaloid extracted from Rauwolfia vomitoria, Rauwolfia obscura and Vinca rosea, as well as from other Apocyanaceae. It belongs to the group of quaternary beta-carbolines. Methods of extraction of this alkaloid have been described by M. Beljanski and J. Bugiel in French Patent Publication FR-A-78 30663 and European Patent Publication EP-A-0 059 817, whose content is incorporated herein by reference. It has also been shown that this alkaloid has a very vivid pale blue fluorescence, which on spectrophotometric analysis corresponds to two absorption maxima, at 252 and 308 nm, respectively, which are characteristic of alstonine and of its isomer serpentine.
It has also been shown, notably in the above-mentioned documents, that alstonine has anti-cancerous properties. These properties are now well substantiated on mouse YC8 lymphoma ascites cells; on Ehrlich "ascites" tumor cells in mice; on type KB, HELA, HEPII and L cancer cell cultures; and on cells originating from nephroblastoma, neuroblastoma or teratoma, all of which are human tumors, primary cultures of which alstonine was found to destroy completely within 24 to 48 hours. By way of comparison, in order to firmly establish the selectivity of action of alstonine against cancerous cells, the same alstonine dose (200 mcg per ml of culture) was applied to primary cultures of monkey kidney cells or to Vero or Circ-type cell lines; in these cases, it produced no cellular destruction within 8 days. This proved the specific interaction of alstonine with the DNA of tumor cells, whereas the DNA of normal cells was not affected by this alkaloid.
However, it was still difficult to have good control of an antitumor treatment using such an alkaloid and/or to establish a reliable diagnosis of a patient's condition in the preoperative, peroperative and post-operative stages. Hence, there was a need for a means for the evaluation of tumoral diseases in order to facilitate the diagnosis of cancerous lesions and of metastases, and to improve their treatment.
SUMMARY OF THE INVENTION
It has now been unexpectedly found that alstonine itself, can be used to obtain a selective diagnosis of tumoral diseases both with regard to their location and to provide an estimate of their size. It has also been found that a similar alstonine composition is useful in cytogenetics. Thus, in accordance with the invention, a method is provided for the detection of tumor cells or of chromosomal aberrations in a tissue sample, comprising treating the tissue sample with a composition comprising alstonine or an isomer thereof, irradiating the treated tissue sample with light having a wavelength effective to induce a fluorescent emission at about 446 nm from alstonine absorbed into tumor cells or cells having a chromosomal aberration; and detecting any fluorescence from the tissue sample. This method can be used for both in vivo and in vitro diagnoses.
The invention also relates to kits for performing cytodiagnostic evaluations on tissue samples in accordance with the method of the invention. Such a kit includes
(a) a composition comprising alstonine or an isomer thereof at a concentration effective to impart measurable fluorescence to tumor cells or to cells having chromosomal aberrations, and
(b) an incubation support adapted for incubation of the tissue sample in the composition.
The invention further relates to an article of manufacture comprising a packaging material and a cytodiagnostic agent contained within said packaging material, wherein said cytodiagnostic agent comprises alstonine or an isomer thereof at a concentration effective to impart
REFERENCES:
patent: 4160019 (1979-07-01), Bjorklund
patent: 4755684 (1988-07-01), Leuirer et al.
patent: 5015463 (1991-05-01), Dougherty et al.
patent: 5283255 (1994-02-01), Levy et al.
Belzanski et al, Exp. Cell Biol, vol. 50, pp. 79-87 (1982).
Belzanski et al. Oncology, vol. 43, pp. 198-203 (1986).
Beljanski et al., "Three alkaloids as selective destroyers of the proliferative capacity of cancer cells", IRCS Medical Science 12: 587-588 (1984).
Beljanski et al., "Three alkaloids as Selective Destroyers of Cancer Cells in Mice", Oncology 43: 198-203 (1986).
Beljanski et al., "Selective Inhibition of in vitro synthesis of Cancer DNA by alkaloids of the .beta.-Carboline Class", Exp. Cell Biol. 50:79-87 (1982).
Mohamed Abdel A.
Weimar Elizabeth C.
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