EP2285420A2 - Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer - Google Patents

Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer

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Publication number
EP2285420A2
EP2285420A2 EP09738115A EP09738115A EP2285420A2 EP 2285420 A2 EP2285420 A2 EP 2285420A2 EP 09738115 A EP09738115 A EP 09738115A EP 09738115 A EP09738115 A EP 09738115A EP 2285420 A2 EP2285420 A2 EP 2285420A2
Authority
EP
European Patent Office
Prior art keywords
cancer
cells
poly
allylamine
cancer cells
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Withdrawn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
EP09738115A
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German (de)
English (en)
Inventor
Silke Krol
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.)
Consorzio Per Il Centro Di Biomedicina Molecolare SCRL
Original Assignee
Consorzio Per Il Centro Di Biomedicina Molecolare SCRL
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Filing date
Publication date
Application filed by Consorzio Per Il Centro Di Biomedicina Molecolare SCRL filed Critical Consorzio Per Il Centro Di Biomedicina Molecolare SCRL
Priority to EP09738115A priority Critical patent/EP2285420A2/fr
Publication of EP2285420A2 publication Critical patent/EP2285420A2/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Withdrawn legal-status Critical Current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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the medical field, in particular to the treatment and diagnosis of cancer.
  • the present invention relates to the use of poly(allylamine) and its pharmaceutically acceptable salt as anticancer agents and as diagnostic agents for the detection and/or treatment of cancer cells in body tissues, as well as theranostic agent for the same purpose, a method fro treating and diagnosing cancer and an apparatus for extracorporeal treatment of blood.
  • Leukemia is a broad term covering a spectrum of diseases, which are clinically and pathologically split into acute and chronic forms.
  • Acute leukemia is characterized by the rapid increase of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. Immediate treatment is required in acute leukemias due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body.
  • Chronic leukemia is distinguished by the excessive build up of relatively mature, but still abnormal, blood cells. Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy.
  • the diseases are classified into lymphoblastic or lymphocytic leukemias, which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form lymphocytes, and myeloid or myelogenous leukemias, which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form red blood cells, some types of white cells, and platelets.
  • Lymphocytic leukemia Acute lymphocytic leukemia Chronic lymphocytic leukemia
  • ALL (or “lymphoblastic" (ALL) (CLL)
  • ALL Acute Lymphocytic Leukemia
  • Treatment for acute leukemia can include chemotherapy, steroids, radiation therapy, intensive combined treatments (including bone marrow or stem cell transplants), and growth factors.
  • CLL Chronic Lymphocytic Leukemia
  • Refractory CLL is a disease that no longer responds favourably to treatment. In this case more aggressive therapies are considered.
  • CLL is probably incurable by present treatments. But, notably, a large group of CLL patients do not require therapy.
  • AML Acute Myelogenous Leukemia
  • Treatment of AML consists primarily of chemotherapy
  • CML Chronic Myelogenous Leukemia
  • Chronic phase CML is treated with inhibitors of tyrosine kinase
  • Blast crisis carries all the symptoms and characteristics of either acute myelogenous leukemia or acute lymphoblastic leukemia, and has a very high mortality rate. This stage can most effectively be treated by a bone marrow transplant after high-dose chemotherapy.
  • HCL Hairy Cell Leukemia
  • Hairy cell leukemia is an incurable, indolent blood disorder in which mutated, partly matured B cells accumulate in the bone marrow.
  • Cancer therapy is still a difficult task for the person expert in this field. Other than surgery, cancer therapy uses complex therapeutic protocols with anticancer drugs and combinations of said drugs and/or radiotherapy. Cancer therapy is mostly affected by severe side effects. Another strongly felt need is a medicament for treating cancer or a medicament for supplementing or enhancing or integrating cancer therapy, which is easy to manage and has reduced side effects.
  • SIGURDSON C, et al. Prion Strain Discrimination Using Luminescent Conjugated Polymers. Nature Methods. 2007, vol.42, no.12, p.1023-1030. disclose the use of luminescent conjugated polymers for characterizing prion strains.
  • YEUNG T., et al. Membrane Phosphatidylserine Regulates Surface Charge and Protein Localization. Science. 2008, vol.319, p.210-213. develop a biosensor to study the subcellular distribution of phosphatidylserine.
  • Some methods are based on the determination of gene expression (WO 2008/19872 A (RICHTER, GUNTHER) 21.02.2008 , WO 2006/89233 A (WYETH) 26.08.2006 , JP 2007244377 A (OKAYAMA UNIV) 27.09.2007 ) or molecular marker determination (US 2007287163 A (GEUIJEN ET AL.) 13.12.2007 , US 2007264261 A (NUVELO INC.) 15.11.2007.
  • cancer stem cells are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease.
  • poly(allylamine), a polyelectrolyte bearing a positive net charge, and its pharmaceutically acceptable salts selectively enters cancer cells or cells in the process of malignant differentiation while it is excluded from healthy cells.
  • poly(allylamine) does not enter healthy cells even upon exposition of said cells for 24 h.
  • the selective uptake of the poly(allylamine) according to the present invention provides a fast and reliable diagnostic tool, which can advantageously be applied to body samples without complex sample preparation being necessary.
  • the poly(allylamine) according to the present invention is also capable of selectively killing the tumour cell.
  • Another object of the present invention is a poly(allylamine) having a positive net charge for use as diagnostic agent for cancer.
  • the present invention provides the use of the above poly(allylamine) as theranostic agents.
  • the concept of theranostic is well-known in the art and is intended as treatment strategy comprising a diagnostic test that identifies patients most likely to be helped or harmed by a new medication, and targeted drug therapy based on the test results.
  • the test results are used to tailor treatment, usually with a drug that targets a particular gene or protein (The Engineer, 2004, 18 (16):38).
  • Another object of the present invention is the above poly(allylamine), which is labelled with a detectable label.
  • Another object of the present invention is poly(allylamine) as diagnostic, therapeutic and theranostic agent for a cancer which comprises at least one population of cancer cells selected from the group consisting of non-adhesive cancer cells, cancer stem cells, undifferentiated and less differentiated cancer cells.
  • cancer are leukaemia, melanoma, liver cancer, breast cancer, ovarian cancer, glioblastoma.
  • Another object of the present invention is a poly(allylamine) or a pharmaceutically acceptable salt thereof for use as diagnostic agent for revealing cancer cells in an isolated tissue of a subject.
  • Another object of the present invention is an ex vivo method for treating a blood sample containing cancer cells from a subject affected by cancer comprising:
  • An apparatus for extracorporeal treatment of blood characterized in that the part of said apparatus contacting the blood for treatment comprises poly(allylamine) or a pharmaceutically acceptable salt thereof is a further object of the present invention.
  • One of the main advantages of the present invention is to provide a theranostic agent which is both fast in its activity and selective towards tumor cells with respect to normal cells. From therapeutically point of view, selectivity is extremely important for safety and compliance of the drug. From diagnostic point of view, selectivity and fast activity is important for reliability of diagnosis and rapidity of response.
  • Another advantage of the present invention is provided by the activity on cancer stem cells and undifferentiated/low differentiated cancer cells, which provides more effective cancer treatment and also the possibility of earlier diagnosis.
  • Still further advantages of the present invention are in a method of treating cancer, in a method of diagnosing cancer and in a method of diagnosis and treatment of cancer, providing polyallylamine as theranostic agent.
  • the present invention can be carried out by treating patient's blood in an extracorporeal process (ex vivo) in an equipment containing polyallylamine according to the present invention.
  • Figure 1 PAH labelled with a fluorophore. Amount of stained cells (HL60, lymphocytes of leukemic and healthy person) after 15 min of incubation.
  • Figure 2 number of living HL60 cells against incubation time with or without FITC-PAH treatment.
  • Figure 3 Graphical representation of MTT test for cell viability vs. polyamine (polyallylamine, polyethylenimine, spermine) concentration for leukemic HL 60, Jurkat cells and normal MNC.
  • polyamine polyallylamine, polyethylenimine, spermine
  • a first object of the present invention is poly(allylamine) or a pharmaceutically acceptable salt thereof for use as antitumor agent.
  • the poly(allylamine) is labelled with a detectable label.
  • the detectable label is compatible with administration in a living subject, without producing substantial damage to said subject.
  • the poly(allylamine) according to the present invention is for use for treating a cancer comprising at least one population of cancer cells selected from the group consisting of non-adhesive cancer cells, cancer stem cells, solid tumors comprising undifferentiated and less differentiated cancer cells.
  • said cancer is selected from the group consisting of leukaemia, melanoma, liver cancer, breast cancer, ovarian cancer and glioblastoma.
  • the poly(allylamine) or a pharmaceutically acceptable salt thereof is for use as diagnostic agent for revealing cancer cells in an isolated tissue of a subject.
  • tissue and samples easily obtainable from a subject are preferred, for example, blood, sputum, urine.
  • said cancer comprises at least one population of cancer cells selected from the group consisting of non-adhesive cancer cells, cancer stem cells, solid tumors with undifferentiated and less differentiated cancer cells.
  • Another object of the present invention is an ex vivo method for treating a blood sample containing cancer cells from a subject affected by cancer comprising: a. contacting said blood sample with a blood purification system whereby cancer cells contained in said blood sample are contacted with poly(allylamine) or a pharmaceutically salt thereof, whereby said poly(allylamine) kills said cancer cells, b. removing unbound poly(allylamine) from the blood sample, to provide a purified blood sample.
  • the present invention comprises also the pharmaceutically acceptable salts of poly(allylamine).
  • a preferred example is poly(allylamine hydrochloride) (PAH).
  • the poly(allylamine) of the present invention when used as a medicament, must be admitted for administration to a patient, being human or animal.
  • poly(allylamine) and its pharmaceutically acceptable salts according to the present invention are commercially available via common suppliers or can be prepared in the laboratory according to common general knowledge of those of ordinary skill in the art.
  • the poly(allylamine) is labelled with a detectable label.
  • the detectable label will bind to the amine group of the polyelectrolyte.
  • the detectable label will be a fluorophore or a chromophore.
  • any sample from a subject suspected to suffer cancer disease or in need to confirm to suffer such a disease is suitable for the diagnosis according to the present invention.
  • Blood is the preferred sample because it is the easiest to obtain and it stains cancer cells wherever found.
  • cancer cells are detected in blood samples of leukemia patients (bone marrow affected) but also in the blood of a patient with diagnosed melanoma (skin cancer) with the risk of metastasis.
  • the method of diagnosis according to the present invention provides contacting the body sample with the poly(allylamine) herein disclosed and revealing the irreversible adhesion of said poly(allylamine) to the cancer cell or the internalization of said poly(allylamine) into said cancer cell.
  • a preferred embodiment provides the detection of cancer cells in blood samples.
  • cancer cells to be detected are non-adhesive cancer cells or invading cancer cells.
  • cancer cells to be detected are leukemia cells or invading cancer cells (detectable in blood because they leave the primary tumour and settle somewhere, causing metastasis).
  • the method of the invention is very convenient, since only a fast centrifugation is needed to separate the lymphocytes.
  • Other preferred embodiments relate to the detection of cancer cells from melanoma, liver cancer, breast cancer, ovarian cancer, and glioblastoma.
  • a particular embodiment of the invention relates to the detection of metastatic cells. Detecting said cells in body circulation, for example blood or lymph circle is of critical importance for the management and cure of cancer disease.
  • the present invention discloses a method for treating cancer comprising: a. obtaining a blood sample from a subject affected by said cancer, b. contacting said blood sample with a blood purification system whereby cancer cells contained in said blood sample are contacted with a solution of a poly(allylamine) according to the present invention, whereby said poly(allylamine) enters said cancer cells, c. removing unbound poly(allylamine), to provide a purified blood sample, d. returning said blood sample to said subject.
  • Another embodiment of the present invention provides an ex vivo method for treating a blood sample containing cancer cells from a subject affected by cancer comprising: a. contacting said blood sample with a blood purification system whereby cancer cells contained in said blood sample are contacted with poly(allylamine) or a pharmaceutically salt thereof, whereby said poly(allylamine) kills said cancer cells, b. removing unbound poly(allylamine), to provide a purified blood sample.
  • ex vivo is intended according to the general meaning given to this term in the medical field, namely that the sample is isolated from a subject in need to be treated and processed according the above method.
  • the method according to the present invention can be wholly performed outside the body of the subject in need of treatment. After the method is completed, the purified blood is returned to the subject.
  • poly(allylamine) is labelled as disclosed before, for example with a fluorescent label. Separation of cancer cells incorporating the labelled polyallylamine is made with any method known in the art, for example with a flow cytometer.
  • a detectable label can also be used if the cancer cells, incorporating the suitable poly(allylamine), are returned to the subject, in order to perform a follow up of the treatment through imaging or any other technique.
  • a non-toxic label is used, such as carotene or other chromophores.
  • Another object of the present invention is an apparatus for extracorporeal treatment of blood, characterized in that the part of said apparatus contacting the blood for treatment comprises poly(allylamine) or a pharmaceutically acceptable salt thereof.
  • a systemic therapy with poly(allylamine) is excluded because of the risk to damage irreversibly the blood vessel or the myocardium but an ex vivo treatment in a blood purification system with strict control that no poly(allylamine) enters the body after treatment will have a high therapeutic potential.
  • the above ex vivo method is highly preferred.
  • poly(allylamine) shall be administered directly into the tumor mass. This administration system is well-known to the person skilled in this art. Guidance can be found in the general common knowledge, for example The Merck Manual of Diagnosis and Therapy, Remington's Pharmaceutical Sciences, last edition, Mack Publishing and Co.
  • PAH poly(allylamine hydrochloride)
  • PEI poly(ethyleneimine)
  • Sp Spermine
  • MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] MgCb and dimethylsulfoxide (DMSO) were purchased from SIGMA-Aldrich (Milan, Italy) and used as received.
  • Leukemia Leukemia (HL60 and Jurkat, (ATCC CCL-240TM and TIB-152TM) cell lines were continuously maintained in DMEM (Gibco ® ,) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich,), penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), gentamicin (10 ⁇ g/ml) at 5% CO2 and 37°C. The medium was changed every second day and mycoplasma testing was performed to exclude any possible contamination prior performing experiments.
  • DMEM Gibco ® , medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich,), penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), gentamicin (10 ⁇ g/ml) at 5% CO2 and 37°C.
  • FBS heat-inactivated fetal bovine serum
  • penicillin 100 units/m
  • JHH-6 cells JCRB1030 undifferentiated hepatocellular carcinoma
  • HRRB Japan Health Science Research Resources Bank
  • JHH-6 were cultured in Williams E medium (Sigma-Aldrich, Missouri, USA. W4128) with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 2mM L-Glutamine (Euro-clone), and 1 %(v/v) antibiotics (10,000 U/mL penicillin, and 10 mg/mL streptomycin (Euro-clone)).
  • FBS fetal bovine serum
  • Euro-clone 2mM L-Glutamine
  • 1 %(v/v) antibiotics 10,000 U/mL penicillin, and 10 mg/mL streptomycin (Euro-clone)
  • the cells were grown at 37°C in a humidified atmosphere 95% air and 5% CO2 under the conditions described above.
  • MNCs Mononuclear cells
  • FITC-PAH fluorescein isothiocyanate-polyallylamine
  • Imaging was performed with Nikon C1 laser scanning confocal unit (Nikon D- eclipse C1Si, Japan) attached to an inversed fluorescence microscope with a 100x/1.49 oil Apo TIRF objective (Nikon, Japan).
  • HL60 and Jurkat cells (20 x 10 4 cells/per well) were plated in 96 well- plates. The cells were exposed to different final concentrations of PAH (20 ⁇ g/mL, 40 ⁇ g /ml_, 100 ⁇ g /ml_, 200 ⁇ g/mL and 400 ⁇ g/mL), along with cells which received no treatment as negative control and 50 ⁇ l DMSO lysed cells as positive control.
  • PAH 20 ⁇ g/mL, 40 ⁇ g /ml_, 100 ⁇ g /ml_, 200 ⁇ g/mL and 400 ⁇ g/mL
  • PEI poly-(ethylenimine), an apoptosis/necrosis inducer or spermine, a natural polyamine and growth inducer
  • a natural polyamine and growth inducer were also tested in the following concentrations: 200, 400, 600, 800 ⁇ g/mL final concentration in the cell medium for spermine.
  • Mononuclear cells from a healthy donor were incubated with a cell density of 2 x 10 5 cells in the same concentrations of PAH solution as before. The treatment was followed by addition of 0.5 mg/mL of MTT and incubation for 1 h at 37°C. The medium was removed, the cells were lysed and the resulting blue formazan crystals were solved in DMSO.
  • the absorbance of each well was read on a microplate reader (Beckman Coulter LD 400C Luminescence detector) at 570 nm.
  • the absorbance of the untreated controls was taken as 100% survival.
  • the present data represents the mean ⁇ sd of three independent experiments.
  • HL60 (260 x 10 4 cells) and Jurkat (645 x 10 4 ) cells were treated with different concentrations (50, 100, 200, 400 ⁇ g) of the polycation (FITC-PAH) or volumes of gel beads (VCP) for 5 min. Cells were centrifuged twice to remove the excess of the compound, the pellet was recovered and resuspended in physiologic solution. Untreated cells were used as negative control.
  • the intracellular green fluorescence from FITC-PAH was collected by Flow Cytofluorometry using a Becton Dickinson FACSCalibur System, equipped with a single argon-ion laser, through a 530 nm band pass filter in combination with a 570 nm dichroic mirror. A minimum of 10,000 cells was analyzed for each sample.
  • the poly(allylamine) used according to the present invention can enter easily in non-adhesive cancer cells, like in case of leukemia or invading cancer cells, while they do not affect normal blood cells (figure 1).
  • the increased permeability inside the cells is also observable for other cancer cells like melanoma, liver, and breast cancer.
  • the percentage is 1.5 ⁇ 0.7 % in case of HL60 cells and 7 ⁇ 1 % in case of Jurkat cells. It is noteworthy that the treated mononuclear cells isolated from healthy donors show nearly no toxic effect of PAH (survival: 80-100%).
  • the two other tested polyamines, PEI and spermine were exemplarily included in figure 3 with one concentration. It was found that PEI is significantly less cytotoxic in short terms (2 h) to the both leukemic cell lines than PAH and spermine is a growth stimulator increasing the number of cells significantly in a concentration dependent manner. PEI is known to induce apoptosis which is a cell death visible only after several hours or days.
  • FITC-PAH is binding to the plasma membrane and the cytoskeletons, but in some cases also to the nucleus and the DNA. Usually binding the strong negativity of heterochromatin nucleoli region of the nucleus within 2 min incubation. After 5 min the poly(allylamine) is seen mostly in the cytoplasm and the nucleus. After 15 min incubation almost all the nucleus are stained with FITC-PAH. Addition of FITC-PAH under microscope, showed that the poly(allylamine) enters the cells very quickly. Within 30 s poly(allylamine) are crossing the plasma membrane and staining the cytoplasm allowing the visualization of internal cell structure.
  • lymphocytes and monocytes (granulocytic component) of leukemic patient show similar behaviour in terms of poly(allylamine) up take as HL60 cell line. They are stained within 30 s and the polycation bind mostly to the plasma membrane and cytoskeleton. After 5 min, 1 h and 2 h incubation we see the same effect as the HL60 cell line that all the cytoplasm and nucleus is totally stained. In patients only the leukemic lymphocytes were stained, but not the healthy ones.
  • PAH had a selective cytotoxicity effect also on different cancer cell lines and cancer stem cell lines, leaving unaffected the normal cells.
  • PAH has a cytotoxic effect on different cancer cells and cancer stem cells, while it has no effect on normal cells.
  • Potential application [00134] The detection of leukemia cells can be in a fast and disposable test kit with high reliability. In a microchamber the dry fluorescent or even chromophore labelled poly(allylamine) is immobilized in a gel. In contact with a blood sample the poly(allylamine) is solving in the serum and entering in the leukemic cells. Imaging under a microscope will give after 1 min the number of cells in which the polyelectrolyte will be able to enter. The cost is very low because the polycation (PAH) used for the test kit is very cheap.
  • the present invention relates to the medical field, in particular to the diagnosis and cure of cancer.

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Abstract

La présente invention concerne le domaine médical, notamment le traitement et le diagnostic du cancer. Plus particulièrement, la présente invention concerne l’utilisation d’une polyallylamine avec une charge nette positive en tant qu’agent anticancéreux et en tant qu’agent de diagnostic pour la détection et/ou le traitement de cellules cancéreuses dans des tissus corporels. Selon la présente invention, la polyallylamine est utile en tant qu’agent théranostic.
EP09738115A 2008-04-30 2009-04-27 Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer Withdrawn EP2285420A2 (fr)

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EP09738115A EP2285420A2 (fr) 2008-04-30 2009-04-27 Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer

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EP08425304A EP2113257A1 (fr) 2008-04-30 2008-04-30 Polyélectrolyte avec chargement net positif à utiliser en tant que médicament et pour le diagnostic du cancer
PCT/EP2009/055069 WO2009133071A2 (fr) 2008-04-30 2009-04-27 Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer
EP09738115A EP2285420A2 (fr) 2008-04-30 2009-04-27 Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer

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EP09738115A Withdrawn EP2285420A2 (fr) 2008-04-30 2009-04-27 Polyélectrolyte avec une charge nette positive destiné à être utilisé en tant que médicament et diagnostic pour le cancer

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